Transcriptome-Based Identification of Genes Responding to the Organophosphate Pesticide Phosmet in Danio rerio.

Organophosphate pesticides (OPPs) are one of the most widely used insecticides. OPPs exert their neurotoxic effects by inhibiting acetylcholine esterase (AChE). Most of the gross developmental abnormalities observed in OPP-treated fish, on the other hand, may not be explained solely by AChE inhibition. To understand the overall molecular mechanisms involved in OPP toxicity, we used the zebrafish (ZF) model. We exposed ZF embryos to an OPP, phosmet, for 96 h, and then analyzed developmental abnormalities and performed whole transcriptome analysis. Phenotypic abnormalities, such as bradycardia, spine curvature, and growth retardation, were observed in phosmet-treated ZF (PTZF). Whole transcriptome analysis revealed 2190 differentially expressed genes (DEGs), with 822 and 1368 significantly up-and downregulated genes, respectively. System process and sensory and visual perception were among the top biological pathways affected by phosmet toxicity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of metabolic pathways, calcium signaling pathway, regulation of actin cytoskeleton, cardiac muscle contraction, drug metabolism-other enzymes, and phototransduction. Quantitative real-time PCR results of six DEGs agreed with the sequencing data expression profile trend. Our findings provide insights into the consequences of phosmet exposure in ZF, as well as an estimate of the potential risk of OPPs to off-target species.

How varying parameters impact insecticide resistance bioassay: An example on the worldwide invasive pest Drosophila suzukii.

Monitoring pesticide resistance is essential for effective and sustainable agricultural practices. Bioassays are the basis for pesticide-resistance testing, but devising a reliable and reproducible method can be challenging because these tests are carried out on living organisms. Here, we investigated five critical parameters and how they affected the evaluation of resistance to the organophosphate phosmet or the pyrethroid lambda-cyhalothrin using a tarsal-contact protocol on Drosophila suzukii, a worldwide invasive pest. Three of the parameters were related to insect biology: (i) sex, (ii) age of the imago (adult stage) and (iii) genetic diversity of the tested population. The two remaining parameters were linked to the experimental setup: (iv) the number of individuals tested per dose and (v) the duration of exposure to the active ingredient. Results showed that response to insecticide differed depending on sex, males being twice as susceptible to phosmet as females. Age principally affected young females' susceptibility to phosmet, because 0-24 hour-old flies were twice as susceptible as 24-48 hour-old and 72-96 hour-old females. Genetic diversity had no observable effect on resistance levels. The precision and accuracy of the median lethal dose (LD50) were greatly affected by the number of individuals tested per dose with a threshold effect. Finally, optimal duration of exposure to the active ingredient was 24 h, as we found an underestimation of mortality when assessed between 1 and 5 h after exposure to lambda-cyhalothrin. None of the main known point mutations on the para sodium channel gene associated with a knockdown effect were observed. Our study demonstrates the importance of calibrating the various parameters of a bioassay to develop a reliable method. It also provides a valuable and transferable protocol for monitoring D. suzukii resistance worldwide.

Phosmet bioactivation by isoform-specific cytochrome P450s in human hepatic and gut samples and metabolic interaction with chlorpyrifos.

Data on the bioactivation of Phosmet (Pho), a phthalimide-derived organophosphate pesticide (OPT), to the neurotoxic metabolite Phosmet-oxon (PhOx) in human are not available. The characterization of the reaction in single human recombinant CYPs evidenced that the ranking of the intrinsic clearances was: 2C18>2C19>2B6>2C9>1A1>1A2>2D6>3A4>2A6. Considering the average human hepatic content, CYP2C19 contributed for the great majority (60%) at relevant exposure concentrations, while CYP2C9 (33%) and CYP3A4 (31%) were relevant at high substrate concentration. The dose-dependent role of the active isoforms was confirmed in human liver microsomes by using selective CYP inhibitors. This prominent role of CYP2C in oxon formation was not shared by other OPTs. The pre-systemic Pho bioactivation measured in human intestinal microsomes was relevant accounting for » of that measured in the liver showing two reaction phases catalysed by CYP2C and CYP3A4. Phosmet efficiently inhibited CPF bioactivation and detoxication, with Ki values (≈30 µM) relevant to pesticide concentrations achievable in the human liver, while the opposite is unlikely (Ki ≈ 160 µM) at the actual exposure levels, depending on the peculiar isoform-specific Pho bioactivation. Kinetic information in humans can support the development of quantitative in vitro/in vivo extrapolation and in silico models for risk assessment refinement for single and multiple pesticides.

Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production.

Epidemiological data have associated environmental organophosphate insecticide (OP) exposure during pregnancy with fetal growth deficits. To better understand OP injury that may adversely affect pregnancy, we used the JEG-3 choriocarcinoma cell line, which provide a recognized in vitro model to study placental function. The effects of the OP phosmet (Pm) and chlorpyrifos (Cp) on JEG-3 cells viability, proliferation, cell cycle and inflammatory molecule production were evaluated. Both insecticides affected cellular viability in a concentration- and time-dependent manner, inducing apoptosis and decreasing [(3)H]-thymidine incorporation. However, only Pm reduced DNA synthesis independently of cellular death and decreased the cell percentage at the S-phase. Unlike apoptosis, TNFα production varied with the concentration tested, suggesting that other TNFα independent mechanisms might trigger cell death. No induction of the inflammatory molecule nitric oxide was detected. The mRNA levels of pro-inflammatory IL-6, IL-17 and the anti-inflammatory IL-13 cytokines were differentially modulated. These findings show that Pm and Cp generate a specific toxicity signature, altering cell viability and inducing an inflammatory cytokine profile, suggesting that trophoblasts may represent a possible target for OP adverse effects.

Relevance of an electrochemical process prior to a biological treatment for the removal of an organophosphorous pesticide, phosmet.

The aim of this study was to examine the feasibility of coupling an electrochemical pre-treatment with a biological step in order to degrade phosmet, an organophosphorous pesticide. Preliminary biodegradation experiments showed that the target molecule was not assimilated by activated sludge. The pre-treatment consisted of potentiostatic electrolysis (-1.3 V/SCE) in a flow cell. After only one pass (1 mL min(-1)), cyclic voltammetry with a vitreous carbon electrode showed a total phosmet reduction in neutral medium confirmed by thin layer chromatography, which also highlighted the presence of several by-products. H NMR spectra of the main by-product showed the absence of the aromatic ring, only the phosphorus part of phosmet has been identified and phosmet-oxon, a very toxic derivative, was not formed. Lower toxicity and higher biodegradability characterized the electrolyzed solution, EC(50) value increased from 7% to 58% and BOD(5) value increased from 4 to 9 mg O(2) L(-1) after electrolysis leading to an increase of the BOD(5) on COD ratio from 0.19 to 0.42 (limit of biodegradability, 0.4). These encouraging results were confirmed during activated sludge culture since an almost total mineralization of the electrolyzed solution was recorded (97%), confirming the feasibility of the proposed coupled process.

Impacts of stage-specific acute pesticide exposure on predicted population structure of the soft-shell clam, Mya arenaria.

A combined laboratory and modeling approach was used to assess the impact of selected pesticides on early life stages of the soft-shell clam, Mya arenaria. Clams were exposed for 24h as veligers or pediveligers to the broad-spectrum herbicide hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1h,3h)-dione; Velpar], the phenoxyacetic acid herbicide, 2,4-D (2,4-dichlorophenoxyacetic acid; Agway Super BK 32), or phosmet (Imidan). In addition, juvenile clams were exposed for 24h to 2,4-D and their growth monitored for 21 months. Laboratory experiments indicated veligers were more sensitive to acute pesticide exposure than pediveligers, with 2,4-D exposed veligers exhibiting the lowest survival among all treatments. Relative to controls, juvenile clams exposed to 0.5 ppm 2,4-D had enhanced survival following the initial 3 months of grow out. Juveniles exposed to 0.5, 5 and 10 ppm 2,4-D showed an initial growth delay relative to control clams, but at 21 months post-exposure these clams were significantly larger than control clams. Data from the larval and juvenile exposures were used to generate a stage-specific matrix model to predict the effect of pesticide exposure on clam populations. Impacts on simulated clam populations varied with the pesticide and stage exposed. For example, 2,4-D exposure of veligers and pediveligers significantly reduced predicted recruitment as well as population growth rate compared to controls, but juvenile exposure to 2,4-D did not significantly reduce population growth rate. With the exception of veligers exposed to 10 ppm, hexazinone exposure at the both veliger and pediveliger stages significantly reduced predicted recruitment success compared to 0 ppm controls. Hexazinone exposure also reduced modeled population growth rates, but these reductions were only slight in the pediveliger exposure simulations. Veliger and pediveliger exposure to phosmet reduced modeled population growth rate in a dose-dependent fashion. Changes in modeled population stable stage distributions were also observed when veligers were exposed to any pesticide. These results suggest that both the stage of exposure and the specific toxicant are important in predicting effects of pesticide exposure on soft-shell clam populations, with earlier life stages showing greater sensitivity to the pesticides tested.

Effects of the insecticide phosmet on solitary bee foraging and nesting in orchards of Capitol Reef National Park, Utah.

Capitol Reef National Park, in southcentral Utah, contains 22 small orchards planted with antique fruit varieties by Mormon pioneers beginning over a century ago. The orchards continue to be managed in a pick-and-pay program, which includes spraying with phosmet to suppress codling moth (Cydia pomonella L.). The park is also home to a rich diversity of flowering plants, many of which are rare, bee-pollinated, and have populations within 1 km of the orchards. Over 3 yr, we studied the short-term effects of phosmet spraying on bee populations: (1) foraging on plants within the orchard understory and adjacent to it; and (2) nesting in, and at several distances from, the orchards. We recorded a rich bee fauna (47 taxa) in the orchards and on plants nearby. In 2 yr (2002 and 2004), we found no difference in the number of native bee visits to several species of plants flowering in and near to orchards immediately before and 1 d after spraying. Conversely, our nesting studies using the semidomesticated alfalfa leafcutting bee, Megachile rotundata (F.), showed strong significant declines in the number of adult males, nesting females, and progeny production subsequent to spraying at distances up to 160 m from sprayed orchards where the bees were presumably foraging. We showed that M. rotundata is negatively affected by phosmet spraying and suggest that caution should be exercised in its use in areas where bees are apt to forage.

Chronic toxic neuropathy after organophosphorus poisoning in quails (Coturnix coturnix japponica).

Peripheral neuropathy was induced by the long-term administration of organo-phosphorus compounds (phtalimid/phosmet) in quails (Coturnix coturnix japponica). After 4 weeks, the first symptoms of organophosphorus (OPC) poisoning (apathy, diarrhea) were present. During the second month of a daily administration of the toxic substance using the probe, an apparent clinical autonomic and peripheral neuropathy with ataxia had developed. Toxic disturbance of the nervous system was confirmed by the examination of spinal and cortical somatosensory evoked potentials (SEP) after tibial nerve stimulation. The prolongation of the peripheral conduction time (wave P6 and N9 represent the response from the ischiadic nerve and the entry of the stimulus to spinal cord, respectively) confirmed a peripheral nerve lesion. We suggest that these clinical and electrophysiological changes, displayed by the disturbed nervous system, are caused by either slowing or stoppage of the axonal flow, transport of proteins and other substances, as well as by axon demyelination (Tab. 1, Fig. 1, Ref. 22).

Neurotoxic potential of six organophosphorus compounds in adult hens.

The neurotoxic potential of trichlorfon, diazinon, phosmet, dichlorvos, phosphamidon and coumaphos was evaluated for their ability to inhibit brain neurotoxic esterase (NTE) activity in adult hens. Leptophos was used as a reference neurotoxic agent. All compounds were administered at high single oral doses and the NTE and acetylcholinesterase (AchE) activities were measured at 24 h and 6 w later. With the exception of leptophos, all compounds produced severe cholinergic signs associated with > 80% inhibition of brain AchE at 24 h. On the other hand, brain NTE activity was 86% inhibited by leptophos and to lesser extents by trichlorfon (76%), phosphamidon (74%), coumaphos (70%) and dichlorvos (70%). However, none of the latter compounds produced clinical delayed neurotoxicity as was observed with leptophos. It was concluded that trichlorfon, phosphamidon, coumaphos and dichlorvos are potentially neurotoxic because of their ability to inhibit brain NTE activity, but the extent of inhibition required for development of clinical delayed neurotoxicity (> 80%) is not likely to occur with any of these compounds due to their severe cholinergic activity.

High-dose exposure to systemic phosmet insecticide modifies the phosphatidylinositol anchor on the prion protein: the origins of new variant transmissible spongiform encephalopathies?

Compulsory exposure of the UK bovine to exclusively high biannual doses of a 'systemic' pour-on formulation of an organo-phthalimido-phosphorus warblecide, phosmet, during the 1980s (combined with exposure to the lipid-bound residues of 'bioconcentrated' phosmet recycled back via the intensive feeding of meat and bone meal), initiated the 'new strain' modification of the CNS prion protein (PrP) causing the UK's bovine spongiform encephalopathy (BSE) epidemic. A lipophilic solution of phosmet was poured along the bovine's spinal column, whence it penetrated and concentrated in phospholipids of the CNS membranes, covalently modifying endogenous phosphorylation sites on phosphatidylinositols (PIs) etc., forming a 'toxic membrane bank' of abnormally modified lipids that 'infect' any membrane proteins (such as PrP) that are programmed to conjugate onto them for anchorage to the membrane. Thus, phosmet invokes a primary covalent modification on PrP's PI anchor which, in turn, invokes an overall diverse disturbance upon CNS phosphoinositide second messenger feed back cycle, calcium homeostasis and essential free radicals; thus initiating a self-perpetuating cascade of abnormally phosphorylated PI-PrP that invokes a secondary electrostatic and allosteric disturbance on the main body of PrP impairing tertiary folding. Chaperone stress proteins conjugate onto misfolded PrP blocking its sites of proteolytic cleavage. Fresh epidemiological evidence is presented and experimental evidence referenced that adds support to a multifactorial hypothesis which proposes that BSE is a hitherto unrecognized and previously unmanifested class of subtle chronic phosmet-induced delayed neuro-excitotoxicity in the susceptible bovine.

Mutagenic activity of phosmet, the active component of the organophosphorus insecticide Decemtione EK 20 in Salmonella and Saccharomyces assays.

Phosmet, the active component of the organophosphorus insecticide Decemtione EK 20, was shown to be mutagenic in the standard Ames Salmonella/mammalian microsome assay in the absence and presence of metabolic activation. It appears to be a direct mutagen inducing base substitution mutations (TA100) as well as a weak frameshift mutagen (TA97). This compound was genotoxic in the Saccharomyces cerevisiae D7 strain. It significantly increased reverse mutation, mitotic crossing-over and slightly, but not significantly, increased gene conversion at the highest concentration used.

Decemtione (Imidan)-induced single-strand breaks to human DNA, mutations at the hgprt locus of V79 cells, and morphological transformations of embryo cells.

To study the genotoxic activity of Decemtione (Imidan), this substance was subjected to a series of tests. After preliminary cytotoxicity testing, the capacity of Decemtione to damage human DNA was determined by alkaline elution of DNA and DNA unwinding. Both tests gave positive results, suggesting that Decemtione was able to induce single-strand breaks in DNA. This capacity was higher in the absence and lower in the presence of the S9 fraction. The potential mutagenicity of Decemtione was followed on the basis of its ability to induce resistance to 6-thioguanine in V79 hamster cells. Unlike the induction of single-strand breaks, Decemtione showed, in the absence of the metabolic activation system, a very weak mutagenic effect, which was, however, significantly higher in the presence of the S9 fraction. The ability of the substance to transform diploid cells under in vitro conditions was followed on the basis of morphological transformation of Syrian hamster embryo cells. The results showed that Decemtione, like positive carcinogenes, induced a significant elevation in morphologically transformed colonies of embryo cells. The results suggest a carcinogenic potential of this organophosphate insecticide.

A rapid in vivo bioassay for the carcinogenicity of pesticides.

Eight pesticides were tested in a bioassay based on the induction of preneoplastic lesions in the liver. Rats were given diethylnitrosamine intraperitoneally at 200 mg/kg bw and two weeks later were treated with pesticides for six weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione s-transferase (placental form) -positive foci in the liver with those of controls given diethylnitrosamine alone. Positive results were seen with Chinomethionat, Phosmet and Propiconazole; the results obtained with Captan and Prochloraz were borderline; Benomyl, Daminozide and Folpet gave negative results. Our findings provide enough experimental evidence to indicate that great care should be exercised in the use of these compounds.

Effect of DNOC, Ferbam and Imidan exposure on mouse sperm morphology.

DNOC, Ferbam and Imidan were tested in (C3H X C57BL/6) F1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular was toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.

Phosmet residues in an orchard and adjacent recreational area.

Two cover sprays of phosmet were applied to an orchard adjoining a camping area and a bird sanctuary with a resident goose population. Insecticide residues were monitored on orchard leaves, orchard ground cover, ground cover in the camp-site and along the adjacent lakeshore. Despite attempts to minimize drift, significant spray residues were found outside the target area. Residues on ground cover and leaves were reduced by sprinkler irrigation subsequent to spray application.

On the mechanism of acute toxicity of phosmet.

Rats were given single oral doses of phosmet (LD50 or 3/4 LD50). Changes of hematological parameters were studied in several time intervals; the main interest was paid to disturbances in blood clotting. Both light and electron microscopy were used to evaluate histopathological changes in selected organs (liver, lungs, spleen, stomach). Marked changes could be observed in all blood clotting parameters studied, other findings were not standard. The pathological picture was dominated by endothelial damage to the blood vessels and findings of thrombocytic clots in microcirculation of all organs examined. Together with hemocoagulation changes, they could be considered as reflecting the compensated stage of diffuse intravascular coagulopathy. In addition, damaged alveolar endothelium and interstitial edema could be observed in the lungs together with hepatic steatosis, damaged proximal renal tubuli, release of cellular elements from the splenic pulp and decomposition of megakaryocytes as well as serious exfoliation of the superficial layers of the gastric mucosa. The pathohistological alterations coincided with changes in organ weights.

Comparison of measurement of dialkyl phosphates in milk/urine and blood cholinesterase and insecticide concentrations in goats exposed to the organophosphate insecticide, imidan.

Recognition of exposure to imidan was assessed in goats by dialkyl phosphate concentrations, blood cholinesterase (ChE) determinations, and blood imidan concentrations. Groups of three goats received 5.0 mg imidan/kg/day (low dose) or 10 mg imidan/kg/day (high dose) for 7 days orally. One goat received no imidan and one goat received an acute single dose (200 mg/kg). The urine of all treated goats was examined for the excretory dialkyl phosphates, O,O-dimethyl phosphorodithioate (DMDTP) and O,O-dimethyl phosphorothionate (DMTP). The overall mean DMDTP urinary concentration was 19.1 ppm (10-mg/kg treatment group) and 7.2 ppm (5-mg/kg treatment group). These metabolites rapidly disappeared following removal of the treatment except in those goats clinically affected. Milk contained no identifiable concentrations of dialkyl phosphates. Cholinesterase depression was observed in all imidan-treated goats, and a dose effect was observed. No imidan was detected in whole blood of either the 5- or 10-mg/kg treatment groups. Low blood concentrations (ppb) of imidan were measured in the acute single-dose exposed goat. Both urinary DMDTP and blood ChE provided recognition of imidan exposure. DMDTP, however, was immediately present in urine after exposure and provided stronger support for organophosphate exposure than did blood ChE.