Responses to clomazone and 5-ketoclomazone by Echinochloa phyllopogon resistant to multiple herbicides in Californian rice fields.

BACKGROUND: Late watergrass [Echinochloa phyllopogon (Stapf.) Koss.] is a major weed of Californian rice that has evolved P450-mediated metabolic resistance to multiple herbicides. Resistant (R) populations are also poorly controlled by the recently introduced herbicide clomazone. The authors assessed whether this cross-resistance was also P450 mediated, and whether R plants also had reduced sensitivity to photooxidation. Understanding mechanism(s) of resistance facilitates the design of herbicide management strategies to delay resistance evolution. RESULTS: Ratios (R/S) of R to susceptible (S) GR(50) were near 2.0. [(14)C]Clomazone uptake was similar in R and S plants. Clomazone and its metabolite 5-ketoclomazone reduced chlorophyll and carotenoids in S more than in R plants. The P450 inhibitors disulfoton and 1-aminobenzo-triazole (ABT) safened clomazone in R and S plants. Disulfoton safened 5-ketoclomazone only in S plants, while ABT synergized 5-ketoclomazone mostly against S plants. Paraquat was more toxic in S than in R plants. CONCLUSION: Cross-resistance to clomazone explains failures to control R plants in rice fields, and safening by P450 inhibitors suggests that oxidative activation of clomazone is needed for toxicity to E. phyllopogon. Clomazone resistance requires mitigation of 5-ketoclomazone toxicity, but P450 detoxification may not significantly confer resistance, as P450 inhibitors poorly synergized 5-ketoclopmazone in R plants. Responses to paraquat suggest research on mechanisms to mitigate photooxidation in R and S plants is needed.

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters.

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.

Insecticide use and organophosphate resistance in the coffee leaf miner Leucoptera coffeella (Lepidoptera: Lyonetiidae).

Increasing rates of insecticide use against the coffee leaf minerLeucoptera coffeella(Guérin-Méneville) and field reports on insecticide resistance led to an investigation of the possible occurrence of resistance of this species to some of the oldest insecticides used against it in Brazil: chlorpyrifos, disulfoton, ethion and methyl parathion. Insect populations were collected from ten sites in the state of Minas Gerais, Brazil and these populations were subjected to discriminating concentrations established from insecticide LC99s estimated for a susceptible standard population. Eight of the field-collected populations showed resistance to disulfoton, five showed resistance to ethion, four showed resistance to methyl parathion, and one showed resistance to chlorpyrifos. The frequency of resistant individuals in each population ranged from 10 to 93% for disulfoton, 53 to 75% for ethion, 23 to 76% for methyl parathion, and the frequency of resistant individuals in the chlorpyrifos resistant population was 35%. A higher frequency of individuals resistant to chlorpyrifos, disulfoton and ethion was associated with greater use of insecticides, especially other organophosphates. This finding suggests that cross-selection, mainly between organophosphates, played a major role in the evolution of insecticide resistance in Brazilian populations of L. coffeella. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide and triphenyl phosphate) suggested that cytochrome P450-dependent monooxygenases may play a major role in resistance with minor involvement of esterases and glutathione S-transferases.

Effects of organophosphate exposure on muscarinic acetylcholine receptor subtype mRNA levels in the adult rat.

Repeated exposure to organophosphorus (OP) insecticides results in a decrease of muscarinic acetylcholine receptors (MRs) in the central nervous system. OP-induced MR down-regulation in vivo is modeled by chronic in vitro exposure to muscarinic agonists. Many studies, both in vivo and in vitro, indicate that the treatment-induced decrease in MR number is accompanied by a decrease in the mRNA levels of specific MR subtypes. In this study, the in vivo effects of OP exposure on the mRNA levels of three MR subtypes (m1, m2, and m3) were examined in brain tissue and in peripheral mononuclear cells, which express the m3 subtype. Adult male Sprague-Dawley rats were orally administered disulfoton (2 mg/kg/day) for 14 days, and a subset of exposed animals was allowed to recover for 28 days. This treatment caused a 28% and 81% decrease, respectively, in [3H]-QNB binding and acetylcholinesterase activity in the cortex, similar to that observed in previous studies; after recovery, these levels had returned to 99% and 90%, respectively, of controls. There was a significant decrease in m1 mRNA levels in hippocampus (23%) after disulfoton treatment, while no change was observed in the cortex, corpus striatum, medulla, or cerebellum. The m2 subtype mRNA was significantly decreased in both hippocampus (24%) and medulla (19%), but not in cortex, striatum, or cerebellum. m3 mRNA levels were significantly decreased in cortex (10%), but no change was observed in hippocampus, medulla, cerebellum, or lymphocytes. After recovery, no differences in m1 or m3 mRNA levels were observed in any tissue examined, whereas the decrease in m2 mRNA in the hippocampus remained significant (29%). These results indicate that OP exposure can differentially regulate mRNA levels for MR subtypes in different brain areas, and suggest that m2 muscarinic receptors in the hippocampus are most affected by this treatment.

Carbachol- and norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition.

Activation of cholinergic muscarinic receptors leads to several biochemical events including an increased turnover of phosphoinositides. In this study we have investigated whether repeated administration of the organophosphorus insecticide disulfoton, known to cause the development of tolerance to this compound, would affect phosphoinositide metabolism in rat brain. Basal and carbachol-stimulated phosphoinositide metabolism were measured in cerebral cortex slices, by measuring the accumulation of inositol phosphates (InsPs) in the presence of lithium. In control animals carbachol caused a 600% increase in InsPs accumulation with an EC50 of 100 microM. Maximal effect occurred with a LiCl concentration of 7.5 mM and required the presence of calcium. Administration of disulfoton for 10 days (2 mg/kg/day by gavage), decreased the number of muscarinic receptors in cortex from 1.1 to 0.7 pmol/mg of protein without changing the affinity of the receptors (both measured by binding of [3H]quinuclidinyl benzilate). Acetylcholinesterase was inhibited by 85%. Basal InsPs accumulation was unchanged in disulfoton-treated rats, whereas carbachol-stimulated InsPs accumulation decreased by 18%. No changes of norepinephrine-stimulated InsPs formation and of alpha-1 adrenoceptors were present in cortices from disulfoton-treated rats. Recovery of muscarinic receptor binding and carbachol-stimulated InsPs accumulation occurred at a similar rate and was completed 2 to 3 weeks after the end of the treatment, whereas acetylcholinesterase activity was still 38% inhibited 3 weeks later. These results support the hypothesis that a functional adaptation of muscarinic receptors is involved in the development of tolerance to organophosphates.

Cholinergic and opiate involvement in the antinociceptive effect of diisopropylfluorophosphate.

The organophosphate diisopropylfluorophosphate (DFP; 3 or 6 mg/kg, IP) caused a dose-related antinociception in mice which was antagonized by the muscarinic antagonist scopolamine. The opiate antagonist naloxone antagonized the antinociceptive effect of the highest dose of DFP, but did not affect the antinociception caused by 3 mg/kg DFP. Twenty-four hours after the administration of DFP, reaction time in animals which received a 3 mg/kg dose did not differ from control. However, reaction time was still significantly higher than control in mice administered 6 mg/kg DFP twenty-four hours earlier. This residual antinociception was antagonized by naloxone but not by scopolamine, suggesting that it was opioid in nature. These results suggest that antinociception induced by a low dose of DFP is primarily due to a cholinergic mechanism, while higher doses appear to affect also the opiate system. Since we have previously shown that DFP (6 mg/kg) increases met-enkephalin levels in brain, it is possible that high doses of DFP might interfere with enkephalin metabolizing enzymes. This conclusion cannot be extended to the organophosphate disulfoton, whose antinociception, even at high doses, appears to involve only an interaction with the cholinergic system.

Chronic administration of an organophosphorus insecticide to rats alters cholinergic muscarinic receptors in the pancreas.

Male rats were treated for 10 days with the organophosphorus insecticide, acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl] phosphorodithioate (disulfoton, 2 mg/kg/day by gavage). At the end of the treatment, binding of [3H]quinuclidinyl benzilate ( [3H]QNB) to cholinergic muscarinic receptors and cholinesterase (ChE) activity were assayed in the pancreas. Functional activity of pancreatic muscarinic receptor was investigated by determining carbachol-stimulated secretion of alpha-amylase in vitro. ChE activity and [3H]QNB binding were significantly decreased in the pancreas from disulfoton-treated rats. The alteration of [3H]QNB binding was due to a decrease in muscarinic receptor density with no change in the affinity. Basal secretion of amylase from pancreas in vitro was not altered, but carbachol-stimulated secretion was decreased. The effect appeared to be specific since pancreozymin was able to induce the same amylase release from pancreases of control and treated rats. The results suggest that repeated exposures to sublethal doses of an organophosphorus insecticide lead to a biochemical and functional alteration of cholinergic muscarinic receptors in the pancreas.

[3H]Nicotine binding in rat brain: alteration after chronic acetylcholinesterase inhibition.

(+/-)-[3H]Nicotine binds specifically to rat brain membranes. The binding is stereospecific, (+)-nicotine being 57 times less potent than (-)-nicotine in displacing labeled (+/-)-nicotine. Saturation binding experiments revealed the presence of two binding sites with dissociation constant (Kd) values of 23.7 and 590 nM, and binding site density (Bmax) values of 76 and 646 fmol/mg of protein, respectively. The substrate specificity of the binding site suggests that it represents the nicotinic cholinergic receptor. [3H] Nicotine binding was found to be highest in the hypothalamus and hippocampus and lowest in the cerebellum. Chronic treatment with the acetylcholinesterase inhibitor disulfoton (2 mg/kg/day for 10 days) decreased the number of cholinergic muscarinic and nicotinic binding sites in rat brain. Moreover, the antinociceptive effect of nicotine was found to be markedly reduced in rats chronically treated with disulfoton.

Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticides.

Male mice treated for 2 weeks with the anticholinesterase insecticide disulfoton (O,O-diethyl S-[2-(ethylthio)-ethyl] phosphorodithioate; 10 mg per kg per day) became tolerant to the hypothermic and antinociceptive effects of disulfoton itself and of oxotremorine, a muscarinic cholinergic agonist. Homogenates of brain and ileum from tolerant animals exhibited reduced binding of the specific muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB). In forebrains of tolerant animals, the number of receptors (Bmax) was decreased 40% with no change in the affinity constant. Acetylcholinesterase (AChE) activity was 15% of control. Forty-eight hours after a single injection of disulfoton (10 mg/kg) mice were more resistant than their controls to the hypothermic and antinociceptive effects of a second administration of the same insecticide and of oxotremorine. Tolerance was not present 96 hr after a single administration of disulfoton. A single injection of disulfoton produced 74, 65 and 27% inhibition of AChE activity after 4, 48 and 96 hr respectively. Four hours after a second injection at 49 and 96 hr, 73 or 72% inhibition was found. [3H]QNB binding of animals treated with a single injection of disulfoton and of controls did not differ at either time point. An increase in the Ki for inhibition of [3H]QNB binding by unlabeled oxotremorine was observed in forebrain from mice killed 48 hr after a single injection of disulfoton, indicating a decreased affinity of the muscarinic receptor for agonists. Binding of [3H]oxotremorine-M was decreased significantly 48 hr after a single injection of disulfoton and after chronic treatment. It is suggested that a differential down-regulation of muscarinic receptors occurs in acute and chronic tolerance, involving agonist and antagonist binding sites and depending on duration of exposure.

Induction of anticholinesterase tolerance in rats with doses of disulfoton that produce no cholinergic signs.

The induction of anticholinesterase tolerance has, in the past, been achieved by using multiple doses of organophosphorus ester insecticides that initially caused cholinergic signs. The purpose of this study was to ascertain whether anticholinesterase tolerance could be induced with doses of an anticholinesterase that produced no overt cholinergic signs. Rats were fed diets containing 0, 7.5, or 20 ppm disulfoton. Animals fed at the 7.5 ppm level were without cholinergic signs throughout the period of feeding. Rats from this group were significantly more resistant to the lethal effect of carbachol than rats given the control diet after 58 and 62 d on the diet. Animals fed 20 ppm disulfoton in the diet initially showed signs that gradually disappeared with time on the diet. Rats fed at this level were more resistant to the lethal effect of carbachol than controls on all challenge dates (30, 45, 58, and 62 d on the diets) and more resistant than animals fed 7.5 ppm only on d 38 and 45. Thus the organophosphorus ester insecticide disulfoton, fed to rats in a dietary concentration that depressed acetylcholinesterase but was insufficient to cause cholinergic signs, induced tolerance to the lethal effects of carbachol. The length of time on an experimental diet before resistance to carbachol could be demonstrated was greater for the experimental group given 7.5 ppm disulfoton than the group given 20 ppm disulfoton in the diet.

Rat hippocampal norepinephrine response to cholinesterase inhibition.

In this study, 13 groups of rats were injected with the insecticide Disulfoton (0-0-diethyl S-2-[(ethylthio) ethyl] phosphorodithioate), sacrificed after various time intervals and levels of hippocampal norepinephrine determined. Results indicate that this cholinesterase inhibiting compound caused a significant decrease (congruent to 14%) in hippocampal norepinephrine within 3 hours of exposure. This reduction was maintained for at least 48 hours. Titers of norepinephrine had returned to control levels when measured 5 days after a single drug administration. Chronic poisoning also caused depressed norepinephrine levels, although not significantly below those observed in acutely treated animals.

Histopathological study of canine optic nerve retina treated by organophosphate pesticide.

The fine structure of pathological changes was examined in the optic nerve and retina of beagle dogs after oral administration by capsule of the organophosphate pesticide ethylthiometon for 2 years. The optic nerve was invariably affected, and the number of myelinated axons markedly reduced. These were substituted for by delicate processes of astrocytes. Changes in the retina were mild and mainly seen in the peripapillary pigment epithelial cells and in the Müller cells of the peripheral retina. These histopathologic findings indicate that long-term administration of ethylthiometon involved both the optic nerce and selected portions of the retina.

A correlation between the dissipation of insecticides and rhizosphere microflora of Abelmoschus esculentus (L.) Moench.

A study to understand the relationship between the dissipation of aldicarb and disulfoton in the rhizosphere region of bhendi and the microbial population revealed a negative correlation between the fungal and Azotobacter population and the residues of both insecticides. However, no definite correlation could be recorded between the populations of bacteria and actinomycetes and the residues of the insecticides.

The effects of chronic ganglion blockade and chronic cholinesterase inhibition on the sensitivity of rabbit stomach muscularis to cholinergic and adrenergic agonists.

The EC50 values, KA values and efficacies (e) of selected agonists have been determined in strips of stomach muscularis from control, chlorisondamine-pretreated and O, O-diethyl S[2(ethylthio)ethyl]phosphodithioate (disulfoton)-pretreated rabbits. Strips from chlorisondamine-pretreated animals were supersensitive to carbachol but normosensitive to phenylephrine; the e but the KA of carbachol was affected by this treatment. The KB of atropine was unchanged. Strips from animals treated with disulfoton were subsensitive to phenylephrine but normosensitive to amidephrine and carbachol; both the e and KA of phenylephrine appeared to be elevated by this pretreatment. The KB of phentolamine remained unchanged. These results indicate that the chlorisondamine-induced supersensitivity is the result of a change beyond the level of the cholinergic receptors. In contradistinction, the subsensitivity to phenylephrine after disulfoton pretreatment may result at least in part, from a qualitative change in the alpha adrenergic receptors in the stomach muscularis.

The effect of Systox on ionic fluxes in excised barley roots.

The influence of various concentrations of the thiol isomer of Systox on ionic fluxes in excised barley roots was investigated. For purposes of comparison, treatments with DNP alone, or in combination with the Systox isomer, were included. Rb and Cl absorption, regardless of the external salt concentrations, were progressively reduced as the Systox concentration in the external solution increased. Treatment with Systox did not result in any appreciable increase in the efflux rates of Rb or Cl ions. DNP treatment enhanced the efflux of Rb but not of Cl. The increased efflux was too small to account for the reduced net uptake. A comparison of individual and combined effects of Systox and DNP on ion absorption indicated an independent action of the two inhibitors.