Inhibitor Report for: Methiocarb

Biochemical mechanisms associated with methiocarb resistance were examined in laboratory-selected and field populations of the western flower thrips, Frankliniella occidentalis (Pergande). Seven populations were examined and they differed in their susceptibility to methiocarb by 30 times. Including the synergists piperonyl butoxide, a cytochrome P-450 monooxygenase inhibitor, or S,S,S-tributylphosphorotrithioate, an esterase inhibitor, in the methiocarb bioassays partially suppressed resistance in the most resistant populations. In vitro assays of general esterase, glutathione S-transferase, and acetylcholinesterase activities showed increased activity in some of the resistant populations and increased activity of the enzymes after methiocarb selection on one of the populations. Assays of acetylcholinesterase sensitivity to inhibition by methiocarb, dichlorvos, and eserine suggested insensitive acetylcholinesterase in two of the resistant populations. These results indicate that methiocarb resistance in F. occidentalis was polyfactorial and involved detoxification and altered target site. None of the biochemical assays showed interpopulation enzymatic differences strongly correlated with the level of methiocarb resistance. The possibilities for developing rapid biochemical diagnostic assays to detect methiocarb resistance in F. occidentalis are discussed.

A strain of western flower thrips (Frankliniella occidentalis) with resistance to methiocarb was collected from a Danish greenhouse for investigation of possible resistance mechanisms. Bioassays with methiocarb showed moderate (10-fold) resistance at LC50 in the greenhouse-collected strain compared to a susceptible laboratory strain. Piperonyl butoxide, a cytochrome P450 monooxygenase inhibitor, showed no synergistic effect on the tolerance level to methiocarb.In vitroassays of enzyme activities showed significantly increased activity of acetylcholinesterase (2.6-fold), general esterases (1.3-fold), and glutathioneS-transferases (1.2-fold) in the resistant strain. Assays of acetylcholinesterase sensitivity to inhibition by methiocarb, dichlorvos, and eserine showed no indications of insensitive acetylcholinesterase in the resistant strain. These results indicated that increased activity of acetylcholinesterase contributed to methiocarb resistance in the resistant strain. The slightly increased activity of general esterases and glutathioneS-transferases may also have contributed to methiocarb resistance.

Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 microM-1.min-1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 microM-1.min-1 for the (A) and (B) enantiomers, respectively).

Methiocarb (MC) is a widely used carbamate pesticide in agriculture and health programs. Although the main molecular mechanism of carbamate toxicity involves acetylcholinesterase inhibition, studies have also implicated the induction of oxidative stress. Therefore, the present study was aimed to evaluate the effect of acute MC exposure on lipid peroxidation, antioxidant defense systems, histological changes in Wistar rats and the protective effect of pretreatment with vitamin E and taurine. A total of 48 rats were randomly divided into six groups. Rats in group I were given corn oil, while those in group III were dosed with vitamin E (100 mg/kg body weight (b.w.)) and in group V were dosed with taurine (50 mg/kg b.w.). Rats in group II were administered with MC only (25 mg/kg b.w., 1/4 of median lethal dose (LD(50))), while those in groups IV and VI were pretreated with vitamin E (100 mg/kg b.w.) and taurine (50 mg/kg b.w.) for 20 days, respectively, and then exposed to MC (25 mg/kg b.w.). The rats administered with MC showed significant increase in the levels of malondialdehyde in the liver and kidney as an index of lipid peroxidation. Levels of glutathione and activities of superoxide dismutase, catalase and glutathione peroxidase were significantly increased, while activity of glutathione reductase remained unchanged in both the tissues after MC treatment. Mild degenerative histological changes were observed in liver tissue, while the changes in kidney tissue were more severe then liver after MC treatment. Pretreatment with vitamin E and taurine resulted in a significant decrease in the lipid peroxidation and alleviating effects on antioxidant defense systems in both the tissues, while protective effects on the histological changes were shown only in kidney when compared with liver. In conclusion, the study has demonstrated that the acute MC exposure in Wistar rats caused oxidative damage on liver and kidney, which were partly ameliorated by the pretreatment of vitamin E and taurine.

BACKGROUND: Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. RESULTS: Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. CONCLUSION: In this work, only DEF shows true synergistic inhibition of WFT esterases.

To compare the toxicity of seven N-methyl carbamates, time course profiles for brain and red blood cell (RBC) cholinesterase (ChE) inhibition were established for each. Adult, male, Long Evans rats (n=4-5 dose group) were dosed orally with either carbaryl (30 mg/kg in corn oil); carbofuran (0.5 mg/kg in corn oil); formetanate HCl (10 mg/kg in water); methomyl (3 mg/kg in water); methiocarb (25 mg/kg in corn oil); oxamyl (1 mg/kg in water); or propoxur (20 mg/kg in corn oil). This level of dosing produced at least 40% brain ChE inhibition. Brain and blood were taken from 0.5 to 24 h after dosing for analysis of ChE activity using two different methods: (1) a radiometric method which limits the amount of reactivation of ChE activity, and (2) a spectrophotometric method (Ellman method using traditional, unmodified conditions) which may encourage reactivation. The time of peak ChE inhibition was similar for all seven N-methyl carbamate pesticides: 0.5-1.0 h after dosing. By 24 h, brain and RBC ChE activity in all animals returned to normal. The spectrophotometric method underestimated ChE inhibition. Moreover, there was a strong, direct correlation between brain and RBC ChE activity (radiometric assay) for all seven compounds combined (r(2)=0.73, slope 1.1), while the spectrophotometric analysis of the same samples showed a poor correlation (r(2)=0.09). For formetanate, propoxur, methomyl, and methiocarb, brain and RBC ChE inhibitions were not different over time, but for carbaryl, carbofuran and oxamyl, the RBC ChE was slightly more inhibited than brain ChE. These data indicate (1) the radiometric method is superior for analyses of ChE activity in tissues from carbamate-treated animals (2) that animals treated with these N-methyl carbamate pesticides are affected rapidly, and recover rapidly, and (3) generally, assessment of RBC ChE is an accurate predictor of brain ChE inhibition for these seven pesticides.

The western flower thrips Frankliniella occidentalis (Pergande) is a very significant pest of a number of different agricultural crops in the south-east of Spain. The importance of thrips as a pest is not due mainly to the direct damage inflicted on the plant, but to the loss in commercial value which occurs as a consequence of the development of dark spots caused by the tomato spotted wilt virus (TSWV) which they transmit. The economic threshold is therefore almost zero, which enhances the problems of resistance management. The present work is part of a global project that attempts to evaluate the status of insecticide resistance in field populations of thrips obtained from several agricultural crops. We have studied, in either individual or pooled insects, some enzyme systems classically related to detoxification of insecticides: esterase and glutathione-S-transferase (GST). The activity of these enzymes from laboratory populations selected with various classes of insecticides has also been measured using several appropriate substrates. An increase in GST mean activity was found in two field-collected strains. Differences in frequency distributions of esterase and GST activities were found for both field-collected strains and for a laboratory strain selected with acrinathrin. These activities were compared with those of a wild-type reference strain.

Biochemical mechanisms associated with methiocarb resistance were examined in laboratory-selected and field populations of the western flower thrips, Frankliniella occidentalis (Pergande). Seven populations were examined and they differed in their susceptibility to methiocarb by 30 times. Including the synergists piperonyl butoxide, a cytochrome P-450 monooxygenase inhibitor, or S,S,S-tributylphosphorotrithioate, an esterase inhibitor, in the methiocarb bioassays partially suppressed resistance in the most resistant populations. In vitro assays of general esterase, glutathione S-transferase, and acetylcholinesterase activities showed increased activity in some of the resistant populations and increased activity of the enzymes after methiocarb selection on one of the populations. Assays of acetylcholinesterase sensitivity to inhibition by methiocarb, dichlorvos, and eserine suggested insensitive acetylcholinesterase in two of the resistant populations. These results indicate that methiocarb resistance in F. occidentalis was polyfactorial and involved detoxification and altered target site. None of the biochemical assays showed interpopulation enzymatic differences strongly correlated with the level of methiocarb resistance. The possibilities for developing rapid biochemical diagnostic assays to detect methiocarb resistance in F. occidentalis are discussed.

A strain of western flower thrips (Frankliniella occidentalis) with resistance to methiocarb was collected from a Danish greenhouse for investigation of possible resistance mechanisms. Bioassays with methiocarb showed moderate (10-fold) resistance at LC50 in the greenhouse-collected strain compared to a susceptible laboratory strain. Piperonyl butoxide, a cytochrome P450 monooxygenase inhibitor, showed no synergistic effect on the tolerance level to methiocarb.In vitroassays of enzyme activities showed significantly increased activity of acetylcholinesterase (2.6-fold), general esterases (1.3-fold), and glutathioneS-transferases (1.2-fold) in the resistant strain. Assays of acetylcholinesterase sensitivity to inhibition by methiocarb, dichlorvos, and eserine showed no indications of insensitive acetylcholinesterase in the resistant strain. These results indicated that increased activity of acetylcholinesterase contributed to methiocarb resistance in the resistant strain. The slightly increased activity of general esterases and glutathioneS-transferases may also have contributed to methiocarb resistance.

Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 microM-1.min-1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 microM-1.min-1 for the (A) and (B) enantiomers, respectively).