Paraoxon resistance

The rat LD qS are 13, 3.6 (oral) and 21, 6.8 (dermal) mg/kg. Parathion is resistant to aqueous hydrolysis, but is hydroly2ed by alkah to form the noninsecticidal diethjlphosphorothioic acid and -nitrophenol. The time required for 50% hydrolysis is 120 d ia a saturated aqueous solution, or 8 h ia a solution of lime water. At temperatures above 130°C, parathion slowly isomerizes to 0,%diethyl 0-(4-nitrophenyl) phosphorothioate [597-88-6] which is much less stable and less effective as an insecticide. Parathion is readily reduced, eg, by bacillus subtilis ia polluted water and ia the mammalian mmen to nontoxic 0,0-diethyl 0-(4-aminophenyl) phosphorothioate, and is oxidized with difficulty to the highly toxic paraoxon [511-45-5] diethyl 4-nitrophenyl phosphate d 1.268, soluble ia water to 2.4 mg/L), rat oral LD q 1.2 mg/kg. 

There are marked species differences in A-esterase activity. Birds have very low, often undetectable, levels of activity in plasma toward paraoxon, diazoxon, pirimi-phos-methyl oxon, and chlorpyrifos oxon (Brealey et al. 1980, Mackness et al. 1987, Walker et al. 1991 Figure 2.10). Mammals have much higher plasma A-esterase activities to all of these substrates. The toxicological implications of this are discussed in Chapter 10. Some species of insects have no measurable A-esterase activity, even in strains that have resistance to OPs (Mackness et al. 1982, Walker 1994). These include the peach potato aphid (Myzus persicae Devonshire 1991) and the... 

Chlordane interachons with other agricultural chemicals are significant and merit additional research. In one study, male Japanese quail (Coturnix japonica) pretreated for 8 weeks with 10 mg chlordane/kg diet had increased resistance to parathion, but not to paraoxon, as judged by cholinesterase activity (Ludke 1977). In another study, northern bobwhites (Colinus virginianus) treated with 10 mg chlordane/kg diet for 10 weeks, followed by endrin stress, had greater accumulations of chlordane in the brain than did birds treated only with chlordane (Ludke 1976). 

Table 10.6 Resistance, esterase activity, and paraoxon degradation for several strains of green peach aphid...

Aphid strain Resistance factor to dimethoate Paraoxon hydrolyzed (pmol/mg aphid/hr)... 

NTE was first identified as the presumptive target of neuropathic OP compounds in the initiation of OPIDN (Johnson, 1970). Its activity in cells and tissues is operationally defined as the enzymatic hydrolysis of the non-physiological substrate, phenyl valerate, which is resistant to inhibition by diethyl 4-nitrophenyl phosphate (paraoxon) and sensitive to inhibition by A.A -diisopropylphosphor-odiamidic fluoride (mipafox) under specified conditions of preincubation with inhibitors and subsequent incubation with substrate (Johnson, 1977 Kayyali et al., 1991 Makhaeva et al., 2007). 

NTE is operationally defined as the esteratlc activity against phenyl phenylvalerate (the preferred substrate), phenyl valerate, or closely related esters that is "resistant" to paraoxon and sensitive to DFP and nlpafox (N,N -dlisopropylphosphorodlaniidlc fluoride) ... 

This enzyme, purified from aphids (24). was very slow in hydrolyzing paraoxon compared to mammalian arylester hydrolases (Table III) however, it was twice as fast in recovery from paraoxon inhibition compared to a porcine carboxylester hydrolase (35) and >300 times faster than monomeric carboxylester hydrolase of rabbit liver (26). No qualitative differences were found in the enzyme, E4, isolated from resistant and susceptible aphids E4 was one of seven electrophoretic forms of hydrolases observed (34). Recovery indicates that resistance is due to both reaction with the insecticide and a very slow turnover, or catalysis. A similar mechanism of was observed with paraoxon in resistant green rice leafhoppers (37). 

In general, resistant acetylcholinesterases are less sensitive as indicated by a smaller bimolecular reaction constant, kj, for phosphorylation of the active site. In our studies of methyl parathion resistant tobacco budworm larvae, lots of ten larval nervous systems were homogenized and kj was determined (22). We observed 25-fold less sensitivity to inhibition to methyl paraoxon in the resistant strain (Table IV). 

NTE is an integral membrane protein in neurons and some non-neural cell types of vertebrates [3,27,32] and its activity depends on lipid contents. It is present in all neurons, but is absent from glia [32], NTE can hydrolyze many esters of carboxylic acids. NTE is conveniently detected in vitro by its ability to catalyse OP-sensitive hydrolysis of an artificial substrate, phenyl valerate. Its activity is operationally defined as phenyl valerate hydrolysis resistant to inhibition by 0,0-diethyl-4-nitrophenyl phosphate (paraoxon, non-neuropathic) and sensitive to inhibition by NJ T-diisopropyl phosphorodiamidic fluoride (mipafox, neuropathic), determined under specified conditions [3]. Differential centrifugation of brain homogenates resulted in an enrichment of NTE in microsomal fractions containing elements of endoplasmic reticulum (ER), Goldgi, and plasma membrane [39],... 

Figure 9.2 Devonshire and Sawicki (1979) at Rothamstead Experimental Station, Hertfordshire, U.K., found seven variants of the aphid Myzus persicae with different resistances to parathion. Excess production of a carboxylesterase as a result of one or several gene duplications was found to be the resistance mechanism. High levels of carboxylesterases take paraoxon away from acetylcholinesterase so that aphids become resistant.

The cotton bollworm, Helicoverpa armigera, in Australia is of great economic importance and is cross-resistant to parathion-methyl and profenofos, but not to chlorpyrifos. It has an acetylcholinesterase with low sensitivity to paraoxon-methyl and profenofos, but the sensitivity to chlorpyrifos is unaltered. As Table 9.3 shows, the enzyme of the resistant insects is a little less efficient by having a slightly higher Km. (Km is the substrate concentration at which an enzyme-catalyzed reaction proceeds at one-half its maximum velocity.) This indicates a somewhat less efficient enzyme, but the difference is so slight that it does not cause any reduced fitness for the insects. The amount of and activity of acetylcholinesterase are almost always much higher than strictly necessary. 

The two ceil lines differ in sensitivity to test compounds mou.se cell line much more resistant to carboxvlesterase inhibition hy ail compounds cell line profiles differ for each esterase carboxy[esterase is less sensitive than AChE and NTE to OP-induced inhibition pretreatment of cells with poor inhibitors of carboxvlesterase followed by treatment with strong inhibitors (paraoxon or malaoxon) is additive, suggesting that carboxvlesterase does not decrease OP compounds available for AChE inhibition. 

Cells isolated from adrenal medulla and cultured 2-6 days total earboxyleslerase activities. B-activity (resistant to 40 p..W paraoxon), C-activity (resistant to 40 piW paraoxon plus 250 pAf mipafox), and NTE activity (difference between B and C) were calculated. 

Bovine chromaffin cells in primary culture possess carboxylcstcrase activities, 73% of w-hich is NTE the NTE component is resistant to inhibition by paraoxon and sensitive to mipafox and is localized to the particulate fraction of cell culture homogenates. Results suggest that bovine chromaffin cells may be a useful model for NTE studies because they possess a higher ratio of NTE/carboxylesterase than adrenal medulla tissue homogenates, brain tissue, or nerv e. 

The optimum dosage determined for CIO2 was nearly equal to that of CI2. Paraoxon was the only intermediate oxidative product formed during the oxidation of parathion by CI2 or CIO2 at pH 7.4. Paraoxon is more resistant to oxidation by CI2 or CIO2 as compared with the parent... 

Laskowski et al. (1975) reported ultrastructural changes in the subsynaptic folds that were quite varied, even between muscle fibers from the same diaphragms of rats acutely treated with paraoxon. The fact that some endplates were totally degenerated after 2 days of paraoxon treatment, while others appeared almost normal even after 5 days, indicates that some endplates are more resistant than others. The most consistent change at the endplates was the presence of vesicular structures in the synaptic clefts. Some regions of the subsynaptic... 

Johnson (1969b, 1970) assumed that phosphorylable sites would be serine-containing proteins. Several car-boxylesters were tested to find a selective substrate able to interact in the same site (able to reduce the speed of [ P]DFP labeling on the mipafox binding fraction). Phenyl-phenyl acetate was selected as that substrate. In other studies, PV was observed to be more selective and has been used for decades for testing the target site, the neurotoxic esterase, and was later called the NTE. NTE has been monitored as the PVase activity resistant to 40 pM paraoxon (20 min) ("B" activity) and sensitive to 40 pM paraoxon plus 60-150 pM mipafox ("C" activity), with NTE as the difference between the activity in condition B and condition C. 

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