Inheritance and stability of mevinphos-resistance in Plutella xylostella (L.), with special reference to mutations of acetylcholinesterase 1.

Diamondback moth (Plutella xylostella L.) causes enormous damage on cruciferous vegetables and can rapidly develop resistance to all kinds of insecticides. To effectively manage the insecticide resistance of P. xylostella, an understanding of its inheritance and stability is essential. Here we investigated the phenotypic and genotypic basis of mevinphos resistance by crossing two genetically pure lines of P. xylostella, an SHggt wild-type strain and an SHMTCN resistant strain carrying 892T/T, 971C/C, and 1156T/G (TCN) mutations of the acetylcholinesterase 1 gene (Pxace1). Similar median lethal concentrations and degrees of dominance in the reciprocal cross progeny, and no plateau on the log concentration-probit line of F1 backcross and self-cross progeny, suggest that the mevinphos-resistance in P. xylostella is inherited as an autosomal and incomplete dominant trait governed by more than one gene. In the absence of mevinphos exposure, the resistance ratio and Pxace1 mutation frequency declined concomitantly in the SHMTCN strain. After 20-generation relaxation, the mevinphos resistance decreased from 52- to 6-fold and the Pxace1 mutation frequency of the TCN haplotype pair decreased from 100% to 0%. A good correlation was found between the resistance ratio and TCN frequency within the range of 12.5- to 25-fold resistance. Since there was no TCN haplotype pair detected below a resistance level of 12.5-fold, we speculate that resistance mechanisms other than target site insensitivity may exist. These observations are important for the prediction and management of mevinphos and related organophosphate resistance in field populations of P. xylostella.

Amino acid substitutions and intron polymorphism of acetylcholinesterase1 associated with mevinphos resistance in diamondback moth, Plutella xylostella (L.).

The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field.

Cholinergic-receptor-independent dysfunction of mitochondrial respiratory chain enzymes, reduced mitochondrial transmembrane potential and ATP depletion underlie necrotic cell death induced by the organophosphate poison mevinphos.

Our current understanding of the nature of cell death that is associated with fatal organophosphate poisoning and the underlying cellular mechanisms is surprisingly limited. Taking advantage of the absence in an in vitro system of acetylcholinesterase, the pharmacological target of organophosphate compounds, the present study evaluated the hypothesis that the repertoire of cholinergic receptor-independent cellular events that underlie fatal organophosphate poisoning entails induction of mitochondrial dysfunction, followed by bioenergetic failure that leads to necrotic cell death because of ATP depletion. Pheochromocytoma PC12 cells incubated with the organophosphate pesticide mevinphos (0.4 or 4mumol) for 1 or 3h underwent a dose-related and time-dependent loss of cell viability that was not reversed by muscarinic (atropine) or nicotinic (mecamylamine) blockade. This was accompanied by depressed NADH cytochrome c reductase, succinate cytochrome c reductase or cytochrome c oxidase activity in the mitochondrial respiratory chain, reduced mitochondrial transmembrane potential, decreased ATP concentration, elevated ADP/ATP ratio, increased lactate dehydrogenase release and necrotic cell death. We conclude that Mev induces cholinergic receptor-independent necrotic cell death by depressing the activity of Complexes I to IV in the mitochondrial respiratory chain, eliciting reduction in mitochondrial transmembrane potential, depleting intracellular ATP contents and damaging cell membrane integrity.

Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms.

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague-Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or Nomega-propyl-L-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4'-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or L-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.

Neuroprotective role of heat shock protein 70 in the rostral ventrolateral medulla during acute mevinphos intoxication in the rat.

Heat shock protein (HSP) is a family of highly conserved proteins that respond to stress and participate actively in cytoprotection. Within the HSP family, HSP70 is the major inducible member that confers protection against cell death. This study investigated whether HSP70 plays a neuroprotective role at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone in the medulla oblongata where the organophosphate insecticide mevinphos (Mev) acts to elicit cardiovascular toxicity. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of Mev (960 microg/kg) induced a significant increase in the HSP70 level in the ventrolateral medulla during phase I ('pro-life' phase), and returned to baseline during phase II ('pro-death' phase) Mev intoxication. Compared to artificial cerebrospinal fluid, normal mouse serum (1:20), or sense hsp70 oligonucleotide (50 pmol) pretreatment, microinjection of an anti-HSP70 antiserum (1:20) or an antisense hsp70 oligonucleotide (50 pmol) bilaterally into the RVLM significantly increased mortality, shortened the duration of phase I intoxication and augmented the induced hypotension in rats that received Mev (960 microg/ kg, i.v.). These results suggest that HSP70 induced in the RVLM during Mev intoxication provides neuroprotection against the organophosphate poison via prevention of cardiovascular depression.

De novo synthesis of ubiquitin carboxyl-terminal hydrolase isozyme l1 in rostral ventrolateral medulla is crucial to survival during mevinphos intoxication.

Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) is a deubiquitinating enzyme that is responsible for making ubiquitin, which is required to target proteins for degradation by the ubiquitin-proteasome pathway in neurons, available. We investigated whether UCH-L1 plays a neuroprotective role at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone in the medulla oblongata where the organophosphate insecticide mevinphos (Mev) acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 microg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with bilateral microinjection into the RVLM of a transcription inhibitor, actinomycin D (5 nmol), or a translation inhibitor, cycloheximide (20 nmol). Compared with aCSF or sense oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the "pro-life" phase, blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. These findings suggest that de novo synthesis of UCH-L1, leading to an enhanced disassembly of ubiquitin-protein conjugates in the RVLM, is essential to maintenance of the "pro-life" phase of Mev intoxication via prevention of cardiovascular depression, leading to neuroprotection.

[Importance of reactivation of fosdrin-inhibited acetylcholinesterase in the brain and diaphragm for the in vivo therapeutic effect of oximes in rats poisoned with fosdrin]. / Význam reaktivace fosdrinem inhibované acetylcholinesterázy v mozku a bránici in vivo pro terapeutickou úcinnost oximu u potkana intoxikovaného fosdrinem.

BACKGROUND: The composition of the causal antidotal therapy of subjects, poisoned with organophosphorus insecticides, has not been satisfactorily solved till now in spite of the knowledge of the basic mechanism of action of these toxic substances. The purpose of this study is to evaluate which of currently used or perspective acetylcholinesterase reactivators seems to be the most efficacious to protect poisoned subjects. METHODS AND RESULTS: In experiments on white laboratory rats, the reactivating efficacy of selected acetylcholinesterase reactivators (oximes), administered at equimolar doses, was evaluated in target tissue of toxic effects of organophosphorus insecticides (diaphragm, brain) and compared with their therapeutic efficacy in the case of prophylactic administration with the help of the evaluation of mean efficacy dose of oximes, possible to protect rats exposed to supralethal dose of organophosphorus insecticide fosdrin for 24 hours. Our findings confirm that there are not statistically significant differences in reactivating and therapeutic efficacy of oximes tested against fosdrin in rats. CONCLUSIONS: The perspective acetylcholinesterase reactivators (H oximes) seem to be as suitable as currently used oximes for the therapy of acute poisonings with organophosphorus insecticides although they are not significantly more efficacious than currently used oximes as in the case of the treatment of poisoning with highly toxic organophosphorus compounds.

Synaptosomal adenosine triphosphatase (ATPase) inhibition by organophosphates.

Chicken spinal cord adenosine triphosphatases (both Na+, K+ stimulated and ouabain insensitive) were inhibited by tri-o-tolyl phosphate (TOTP, a neurotoxic organophosphate which is not a cholinesterase inhibitor) and mevinphos (a non-neurotoxic compound but inhibitor of cholinesterases). The inhibition was concentration and time dependent, with an initial rapid drop in activity followed by a gradual exponential decline.

Effects of mevinphos (Phosdrin) on unit discharge patterns in avian hippocampus.

Low doses of the acetylcholinesterase inhibitors, mevinphos and physostigmine, reduce hippocampal inhibitory phenomena, measured in terms of the duration of the poststimulus inhibitory pause seen in individual hippocampal neurones in the pigeon. The cholinergic antagonist, atropine, increased hippocampal inhibition. At near threshold doses, mevinphos could, in part, reverse atropine effects. At higher doses, mevinphos synergized with atropine. In the discussion, it is suggested that any organophosphate or carbamate pesticide and its antidote, atropine, may alter "attention" or "short-term memory" through effects on hippocampus at doses too low to induce grossly detectable peripheral symptomatology. Extreme caution in the use or handling of these compounds by aerial applicators is urged. Since the effects of atropine and mevinphos can synergize, the use of atropine to permit poisoned applicators to resume activity is questioned.

Effects of organophosphate pesticides and other drugs on subcortical mechanisms of visual integration.

Atropine, scopolamine, mevinphos, and eserine selectively block directional sensitivity of visual integrative neurones in the thalamus. Cholinergic drugs that do not penetrate the blood-brain barrier are without effect. The neurones studied are important links in reflex brain systems controlling visual attention and eye movements. The results suggest that any cholinergic drug that can get into the brain will disturb visual functions. Since the changes are qualitative and the system is reflex, the affected individual may be unaware of dysfunction. The resultant dangers to aerial applicator personnel are discussed, particularly with respect to atropine, which is necessary in the therapy of organophosphate and carbamate poisoning but is potentially harmful if self-administered for either pro-phylaxis or treatment.

Some behavioral effects of pesticides: The interaction of mevinphos and atropine in pigeons.

The interaction of the organophosphate mevinphos and atropine was examined in two pigeons performing in a variable interval schedule of reinforcement. When administered separately, both atropine and mevinphos produced a dose-related decrement in responding. The combined exposure to these drugs produced a performance decrement greater than that caused by exposure to each component drug alone. These findings suggest that prophylactic use of atropine may increase the detrimental behavioral effects of organophosphate exposure and that the atropine exposure alone may produce serious behavioral deficits.

The effects of mevinphos on appetitive operant behavior in the gerbil.

The need for study of the effects on performance of non-lethal organophosphate insecticide exposure is founded on many reports of behavioral difficulties in aerial applicators following exposure. In this study, a different pair of gerbils served in each of the following schedules of reinforcement: FR 25, FR 75, DRL 12-sec, DRL 20-sec, and VI 1-min. Baseline performance in these tasks tended to be comparable to that of more common laboratory species, but was more variable in the case of the VI 1-min task. Mevinphos doses of 0.20 mg/kg and above produced observable somatic signs of poisoning and also produced dose-related decrements in performance in FR and VI tasks. Performance in the DRL schedule was affected only at a dose of 0.30 mg/kg. No performance deficits or overt somatic signs of poisoning were present at mevinphos doses of 0.10 mg/kg or lower. These results do not agree with those of an earlier study which decrements in VI performance of pigeons and squirrel monkeys appeared at low mevinphos doses which did not produce overt somatic signs of poisoning. The possibility of variations in mevinphos effect as a function of species and task was discussed.