Inhibitor Report for: Chlorpyrifos-methyl-oxon

The pinewood nematode, Bursaphelenchus xylophilus, causes severe damage to pine species by transmitting the pine wilt disease, and the injection of nematicides, such as emamectin benzonate and milbemectin, is the most common practice to control this pest. However, despite their high efficacy, these macrocyclic lactone nematicides are expensive, limiting their practicability. In an attempt to screen affordable alternative nematicidal agents, we expressed three recombinant acetylcholinesterases (ACEs, EC 3.1.1.7) of B. xylophilus using an in vitro baculovirus expression system and evaluated the effects of 11 organophosphates (OPs) and three carbamates (CBs) on the toxicological properties of the enzymes. Of the three recombinant B. xylophilus ACEs (BxACEs; BxACE-1, BxACE-2 and BxACE-3), BxACE-1 and BxACE-2 were highly inhibited by OPs and CBs, including paraoxon, dichlorvos, chlorpyrifos-oxon, chlorpyrifos-methyl-oxon, mevinphos and carbofuran, as demonstrated by an inhibition assay. In contrast, BxACE-3 was insensitive to most of the pesticides tested, with the exception of mevinphos. BxACE-2 was more sensitive than BxACE-1 and BxACE-3 to most of the OPs, whereas BxACE-1 was more sensitive to the CBs than BxACE-2 and BxACE-3. An in vivo toxicity assay revealed that some compounds that were rarely employed as nematicides exhibited higher toxicities than those chemicals commonly used as nematicides. The inhibition kinetic data and in vivo toxicity assay obtained in this study should provide essential information for the development of OP- and CB-based nematicides against B. xylophilus. The availability of recombinant ACEs will also facilitate the development of an in vitro screening system to develop potential OP- and CB-based nematicides.

Noncatalytic detoxication of the oxons of four phosphorothionate insecticides (parathion, methyl parathion, chlorpyrifos, and chlorpyrifos methyl) by boll weevil aliesterase was determined. All the oxons except methyl paraoxon were potent inhibitors of aliesterase (I50s in 1-100 nMrange). Paraoxon and chlorpyrifosoxon were more potent inhibitors of aliesterase than of acetylcholinesterase, while the reverse was true for methyl paraoxon and chlorpyrifos-methyl-oxon. Boll weevil homogenates (containing EDTA and no Triton X-100, to eliminate possible interference by A-esterase and acetylcholinesterase) significantly increasedI50s of four oxons to bovine brain acetylcholinesterase, indicating detoxication of the oxons by the homogenates. The degree of detoxication (% detoxication/mgweevil/ml) for the oxons was chlorpyrifos-oxon > chlorpyrifos-methyl-oxon > paraoxon > methyl paraoxon, which correlated with weevil aliesterase sensitivity to inhibition by these oxons.In vivotreatments of weevils with any one of three aliesterase inhibitors, DEF (S,S,S-tributyl phosphorotrithioate), DAPDT (S,S-diamyl phenylphosphonodithioate), or BPNxn (n-butyl 4-nitrophenyl phenylphosphonate), all caused an appreciable reduction of thein vitrodetoxication. Using chlorpyrifos-oxon as a representative insecticide, the detoxication by homogenates of DEF-treated weevils was a function of residual aliesterase activity.In vivostudies indicated a trend of an inverse relationship betweenin vivotoxicities and antialiesterase activities of the four phosphorothionate insecticides. Substrate specificity of aliesterase for four esters was alpha-naphthyl propionate > alpha-naphthyl acetate > alpha-naphthyl butyrate > nitrophenyl butyrate. The more toxic insecticides, chlorpyrifos methyl and methyl parathion, showed little or no inhibition of any of the substrate hydrolases while the less toxic chlorpyrifos and parathion and the two aliesterase inhibitors (DEF, BPNxn) all selectively and strongly inhibited aliesterase activities toward nitrophenyl butyrate and alpha-naphthyl butyrate. Theses results suggest that low toxicities of the insecticides may have resulted from higher affinity for specific aliesterases, and their greater synergism by aliesterase inhibitors demonstrated previously is because of selective blocking of the specific aliesterase activities by the synergists. Therefore,in vitroandin vivostudies both demonstrated that aliesterase played an important role in noncatalytic detoxication of organophosphorus insecticides in boll weevils. Acting as an alternative phosphorylation site, aliesterase reduces the concentration of organophosphorus insecticides reaching acetylcholinesterase and, thus, provides a protection to this target. The level of this detoxication depends on relative sensitivities or affinities of the oxons for aliesterase and acetylcholinesterase.

An automated method based on normal-phase LC has been developed for the sample clean-up of mussel extracts prior to gas chromatographic analysis of residues of chlorpyrifos, chlorpyrifos-methyl and their metabolites chlorpyrifos-methyl-oxon and 3,5,6-trichloro-2-pyridinol. Pesticides were extracted by means of a high speed blender using acetonitrile-acetone (90:10, v/v). The extract obtained was filtered and concentrated using rotavapor and the residue was dissolved in hexane. One ml of the hexanoic extract was injected on the silica-gel column, using hexane as mobile phase. Pesticides and metabolites were eluted in fat-free fractions with different mixtures of hexane-ethyl acetate. Diode array detection allowed monitoring on-line the elution of lipids. Purified extracts were analyzed by GC using nitrogen-phosphorus detection for quantitation and MS for confirmatory purposes. The method is fully automated from the injection of the extract to the collection of fractions, which are directly injected into the GC system. In this way, neither further clean-up nor solvent exchange were necessary prior to GC analysis. Recoveries obtained from fortified mussel samples at two concentration levels-100 and 20 ng g-1 for parent pesticides and 200 and 40 ng g-1 for metabolites-were higher than 90%. Limits of detection of the whole procedure of analysis were lower than 1 ng g-1 for parent pesticides and than 10 ng g-1 for metabolites. This method has been successfully applied to bioconcentration studies with mussels exposed to chlorpyrifos. Chlorpyrifos and its metabolic derivative 3,5,6-trichloro-2-pyridinol were detected and confirmed by MS in analyzed samples.