Mixed-function Oxidase Inhibitor Research Articles

Field-evolved pyrethroid resistance in Tychius picirostris (Coleoptera: Curculionidae) populations in Oregon white clover seed crops.

The clover seed weevil, Tychius picirostris Fabricius, a serious pest of white clover, Trifolium repens L., grown for seed in western Oregon, causing feeding damage to flowers and developing seeds. Since 2017, white clover seed producers have anecdotally reported T. picirostris control failures using foliar pyrethroid insecticide applications. This mode of action (MoA) is an important chemical control option for T. picirostris management. To evaluate insecticide resistance selection to pyrethroids (bifenthrin) and other MoAs labeled for T. picirostris management (malathion and chlorantraniliprole), adult populations were collected from 8 commercial white clover grown for seed fields in the Willamette Valley, OR, in 2022 and 2023. Among collected Oregon populations, very high resistance ratios (RR50 = 178.00-725.67) were observed to technical grade bifenthrin and low to high resistance ratios (RR50 = 7.80-32.80) to malathion in surface contact assays compared to a susceptible Canadian field population. Moreover, >2.73 times the labeled rate of formulated product containing bifenthrin as the sole MoA was required to kill >50% of T. picirostris in topical assays. Synergistic assays with a mixed-function oxidase inhibitor, an esterase inhibitor, and a glutathione-S-transferase inhibitor revealed phase I and II detoxification enzymes are present in Oregon T. picirostris populations and confer metabolic resistance to bifenthrin. This is the first report of T. picirostris insecticide resistance selection to pyrethroid and organophosphate insecticides. Results will inform continued monitoring and insecticide resistance management strategies to slow the evolution of T. picirostris insecticide resistance selection in Oregon's white clover seed production.

Joint Action of Two Pesticides and an Oxidase Inhibitor on The Snail Lymnaea Acuminata

Studies were conducted on synergism between an Organophosphate, Nuvan (dichlorvos) mixed with a carbamate, Sevin (carbaryl) in a 1:46 ratio against the snail Lymnaea acuminata. It was found that Sevin enhances the activity of Nuvan and thus the LC values ( LC10, LC50, LC90 ) obtained from this mixture were low as compared to Nuvan or Sevin alone. The acetylcholinesterase (AChE) inhibition was higher in case of Nuvan + Sevin (16.17%) as compared to Nuvan or Sevin (34.7% , 69.1%) alone. When Piperonyl Butoxide (PB), a mixed function oxidase inhibitor was given with Nuvan and Sevin in a 1:46:5 ratio, the LC value decrease still further. It appears that, in the tertiary mixture, while PB reduce the oxidative detoxification of Nuvan and Sevin the carbamate may also be preventing the hydrolysis of Nuvan may tertiary mixture lethal than of its components.

Resistance monitoring to four insecticides and mechanisms of resistance in Lygus lineolaris Palisot de Beauvois (Hemiptera: Miridae) populations of southeastern USA cotton.

Economically damaging infestations of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), have become more frequent in Virginia and North Carolina cotton since 2013 and 2010, respectively. Foliar insecticide use has increased dramatically in response. Efficacy data (LC50 ) are needed to evaluate L. lineolaris susceptibility and resistance levels (RR50 ) to commonly used and recommended insecticides for managing this pest in the southeastern USA. Elevated resistance levels to acephate and bifenthrin were measured in L. lineolaris populations collected from wild and cultivated hosts in Virginia, North Carolina and northern Alabama when compared to a susceptible laboratory population. High levels of bifenthrin resistance were observed in 2018 and 2019. Mixed-function oxidase and esterase (EST) inhibitors, piperonyl butoxide and S,S,S-Tributyl phosphotrithioate, respectively, had a synergistic effect on bifenthrin with resistant populations of L. lineolaris. Bifenthrin-resistant L. lineolaris populations expressed elevated levels of cytochrome P450 (CYP450 ) monooxygenase and general EST activity. Results suggest that insecticide resistance is present in some locations and that CYP450 and EST activity in L. lineolaris contribute to pyrethroid resistance in the southeastern USA. Results can serve as a baseline for continued monitoring of L. lineolaris insecticide resistance and inform insecticide resistance management strategies that help southeastern USA cotton producers to minimize inputs and slow resistance development. © 2020 Society of Chemical Industry.

Re-Visiting Insecticide Resistance Status in Anopheles gambiae from Côte d'Ivoire: A Nation-Wide Informative Survey

Insecticide resistance constitutes a major threat that may undermine current gain in malaria control in most endemic countries. National Malaria Control Programmes (NMCPs) need as much information as possible on the resistance status of malaria vectors and underlying mechanisms in order to implement the most relevant and efficient control strategy. Bioassays, biochemical and molecular analysis were performed on An. gambiae collected in six sentinel sites in Côte d'Ivoire. The sites were selected on the basis of their bioclimatic status and agricultural practices. An. gambiae populations across sites showed high levels of resistance to organochloride, pyrethroid and carbamate insecticides. The kdr and ace-1R mutations were detected in almost all sentinel sites with mosquitoes on the coastal and cotton growing areas mostly affected by these mutations. At almost all sites, the levels of detoxifying enzymes (mixed-function oxidases (MFOs), non-specific esterases (NSE) and glutathione-S-transferases (GSTs)) in An. gambiae populations were significantly higher than the levels found in the susceptible strain Kisumu. Pre-exposure of mosquitoes to PBO, an inhibitor of MFOs and NSEs, significantly increased mortality rates to pyrethroids and carbamates in mosquitoes but resistance in most cases was not fully synergised by PBO, inferring a residual role of additional mechanisms, including kdr and ace-1 site insensitivity. The large distribution of resistance in Côte d'Ivoire raises an important question of whether to continue to deploy pyrethroid-based long-lasting insecticidal nets (LLINs) and insecticide residual spraying (IRS) towards which resistance continues to rise with no guarantee that the level of resistance would not compromise their efficacy. Innovative strategies that combine insecticide and synergists in LLINs or spatially LLIN and an effective non-pyrethroid insecticide for IRS could be in the short term the best practice for the NMCP to manage insecticide resistance in malaria vectors in Côte d'Ivoire and other endemic countries facing resistance.

Evaluation of PermaNet 3.0 a deltamethrin-PBO combination net against Anopheles gambiae and pyrethroid resistant Culex quinquefasciatus mosquitoes: an experimental hut trial in Tanzania

BackgroundCombination mosquito nets incorporating two unrelated insecticides or insecticide plus synergist are designed to control insecticide resistant mosquitoes. PermaNet 3.0 is a long-lasting combination net incorporating deltamethrin on the side panels and a mixture of deltamethrin and synergist piperonyl butoxide (PBO) on the top panel. PBO is an inhibitor of mixed function oxidases implicated in pyrethroid resistance.MethodAn experimental hut trial comparing PermaNet 3.0, PermaNet 2.0 and a conventional deltamethrin-treated net was conducted in NE Tanzania using standard WHOPES procedures. The PermaNet arms included unwashed nets and nets washed 20 times. PermaNet 2.0 is a long-lasting insecticidal net incorporating deltamethrin as a single active.ResultsAgainst pyrethroid susceptible Anopheles gambiae the unwashed PermaNet 3.0 showed no difference to unwashed PermaNet 2.0 in terms of mortality (95% killed), but showed differences in blood-feeding rate (3% blood-fed with PermaNet 3.0 versus 10% with PermaNet 2.0). After 20 washes the two products showed no difference in feeding rate (10% with 3.0 and 9% with 2.0) but showed small differences in mortality (95% with 3.0 and 87% with 2.0). Against pyrethroid resistant Culex quinquefasciatus, mediated by elevated oxidase and kdr mechanisms, the unwashed PermaNet 3.0 killed 48% and PermaNet 2.0 killed 32% but after 20 washes there was no significant difference in mortality between the two products (32% killed by 3.0 and 30% by 2.0). For protecting against Culex PermaNet 3.0 showed no difference to PermaNet 2.0 when either unwashed or after 20 washes; both products were highly protective against biting. Laboratory tunnel bioassays confirmed the loss of biological activity of the PBO/deltamethrin-treated panel after washing.ConclusionBoth PermaNet products were highly effective against susceptible Anopheles gambiae. As a long-lasting net to control or protect against pyrethroid resistant mosquitoes PermaNet 3.0 showed limited improvement over PermaNet 2.0 against Culex quinquefasciatus.

INVESTIGATION OF RESISTANCE MECHANISMS IN SPINOSAD AND ABAMECTIN RESISTANT STRAINS OF COTTON LEAFWORM, Spodoptera littoralis (BOISDUVAL)

The role of mixed function oxidases (MFO), hydrolytic cleavage enzymes and glutathione-mediated reactions in resistance of cotton leaf worm (CLW) Spodoptera littoralis (Boisd.) toward spinosad and abamectin were investigated using spinosad dipping resistant strain (SDRS), spinosad feeding resistant strain (SFRS) and abamectin dipping resistant strain (ADRS) and compared with the parent field strain (PS). Piperonyl butoxide (PB), MDPOC, triphenyl phosphate (TPP) and diethyl maleate (DEM) as mixed function oxidase, esterase and glutathione transferase inhibitors,respectively, were used in this investigation. The effect of cuticle permeability on the two mentioned insecticides was also studied by injecting the fifth instar larvae of S. littoralis with the two insecticides and treating them topically. Results emphasized that no role of cuticle penetration was found as resistance mechanism in the two spinosad resistant strains (SDRS and SFRS). While, cuticle permeability of ADRS was considered a responsible factor for the resistance of cotton leaf worm to abamectin. Values of synergistic ratio (SR) of spinosad in SDRS were 0.89, 1.11, 0.80 and 1.78 with PB, MDPOC, TPP and DEM, respectively. While SR values of spinosad in SFRS with the same corresponding synergists were 1.70, 1.96, 1.22 and 2.05, respectively. No significant differences were found between spinosad toxicity alone or with any of tested synergist in both strains SDRS and SFRS. These results suggest that metabolic detoxification enzymes play very limited role in the resistance mechanism(s) to spinosad in cotton leafworm (CLW). Values of SR in ADRS with the same corresponding synergists were 3.33, 2.29, 3.08 and 3.33, respectively. While SR values in the parent field strain were 1.08, 1.00, 0.97 and 1.03, respectively, with the same corresponding synergists. These results indicated high activity of detoxifying enzymes in ADRS compared with the parent field strain.

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione- S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione- S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione- S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.

Multiple Resistance and Biochemical Mechanisms of Pyridaben Resistance in <I>Tetranychus urticae</I> (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.

Influence of malathion pretreatment on the toxicity of anilofos in male rats: a biochemical interaction study

Toxicity of organophosphates stems mainly from the accumulation of acetylcholine due to inhibition of acetylcholinesterase (AChE). The consequences of excess acetylcholine depend on the events initiated by the interaction of acetylcholine with cholinergic receptors. Lipid peroxidation (LPO) induced by organophosphates also seems to be mediated via cholinergic receptors. Anilofos is a widely used thionoorganophosphate herbicide, while malathion is a thionoorganophosphate insecticide. Thionoorganophosphates undergo mixed function oxidase (MFO)-catalyzed bioactivation to oxons and can induce cholinergic crisis in mammals. Thus, factors (e.g. exposure to certain xenobiotics) which alter the MFO activity, can be assumed to affect the toxicity of these organophosphates. It was investigated in rats if malathion as an inhibitor of MFO can alter the toxicity of anilofos, examining certain biochemical traits in blood, brain and liver. Malathion or anilofos and their combination did not produce any obvious signs of toxicity. Malathion did not alter the anticholinesterase action of anilofos in blood, brain and liver. LPO was increased in erythrocytes, brain and liver with anilofos or malathion and their combination. Production of lipid peroxide in brain of malathion-pretreated rats given anilofos was significantly greater than in rats given anilofos alone. Malathion decreased glutathione (GSH) contents of liver and blood. Glutathione-S-transferase (GST) activity was decreased in the liver with malathion and its combination with anilofos. Total adenosine triphosphatase (ATPase) activity was not affected. Activities of Mg 2+-ATPase and Na +–K +-ATPase were increased in the liver and erythrocytes, respectively, with the pesticide combination. Protein level in plasma was decreased with malathion and its combination with anilofos, but only with the combination in the liver. Results of the study indicate that malathion pretreament may not essentially alter the anticholinesterase action of anilofos, but may enhance anilofos-mediated oxidative damage to rat brain.

Methidathion Resistance Mechanisms in Amblyseius womersleyi Schicha (Acari: Phytoseiidae)

Methidathion resistance mechanisms were studied in the predaceous mite Amblyseius womersleyi . High synergistic ratios (SR = LC 50 without synergist/LC 50 with synergist) were observed for piperonyl butoxide and 2-propynyl 2,3,6-trichlorophenyl ether (both inhibitors of mixed-function oxidases) only in the resistant strain, suggesting that an increase in oxidative metabolism is involved in this resistance. Tests done with the synergists DEM (an inhibitor of GSH transferases and mfo) and DEF (an inhibitor of esterases and GSH transferases) indicate that, to a lesser extent, hydrolytic reactions are also partially involved in this resistance. Insensitive acetylcholinesterase was thought to be a minor factor for resistance. No difference in cuticular permeability of [ 14 C]methidathion was observed between the susceptible and resistant strains. An in vitro degradation study showed that [ 14 C]methidathion was degraded 1.5 times more in the microsomal fraction of the resistant strain than that of the susceptible one. No difference in [ 14 C]methidathion degradation was observed in the soluble fraction between susceptible and resistant strains. From these results, oxidative degradation was thought to have a critical role in methidathion resistance in A. womersleyi .

A Novel Pathway for Hormonally Active Calcitriol

Calcitriol [1α,25(OH)₂D₃], the hormonally active form of vitamin D₃ (D₃) is produced in both renal and extrarenal tissues. Our findings demonstrate that physiological doses of UVB radiation at 300 nm induce the conversion of 7-dehydrocholesterol (7-DHC) via preD₃ and D₃ into calcitriol in the pmol range in epidermal keratinocytes. The hydroxylation of photosynthesized D₃ to calcitriol is strongly suppressed by ketoconazole, a known inhibitor of cytochrome P450 mixed function oxidases. The UVB-induced formation of calcitriol in human skin is demonstrable in vivo by the microdialysis technique. These results suggest that human skin is an autonomous source of hormonally active calcitriol.

Enhancing the margin of selectivity of RPA 201772 inZea mayswith antidotes

The antidotes dichlormid, MON-4660, CGA-154281, R-29148, and MON-13900 were tested in the greenhouse to protectZea maysL. (corn) against RPA 201772 injury. High rates of RPA 201772 injured four Z.mayshybrids &gt; 30%. R-29148 and MON-13900 were the most effective of the five antidotes evaluated. R-29148 applied at rates ⩾ 45 g ha−1provided excellent protection against RPA 201772 injury and also prevented injury toZ. maysfrom diketonitrile, the active metabolite of RPA 201772. In laboratory studies, R-29148 did not alter absorption of14C-RPA 201772 from soil; however, R-29148 significantly enhanced the rate of RPA 201772 metabolism and inactivation inZ. mays.The mixed function oxidase inhibitor piperonyl butoxide (PBO) increased RPA 201772 injury on all hybrids. These results demonstrate thatZ. maystolerance to RPA 201772 can be enhanced with the use of antidotes such as R-29148 and MON-13900, that R-29148 protectsZ. maysfrom RPA 201772 and diketonitrile by the enhancement of metabolism, and that oxidative reactions may be involved in the metabolism of RPA 201772 inZ. mays.

A Novel Bioactivation Mechanism of Phosphoramidate Insecticides

In order to elucidate the activation mechanism of phosphoramidate insecticides, 2,4-dichlorophenyl methylN-alkylphosphoramidates (alkyl: methyl, ethyl, propyl, isoproppyl, and butyl) were chosen as model compounds and compared with isofenphos oxon. Although they showed a potent insecticidal activity against susceptible housefly except for butyl homologue, theirin vitroacetylcholinesterase (AChE) inhibitory activity was very poor.N-Desalkyl compound has been known as an activated form. When bovine erythrocyte AChE inhibition ofN-isopropyl homologue and isofenphos oxon were carried out in rat liver microsomal system, they gave a strong inhibitory activity under the oxidative condition with NADPH, but, in the presence of SKF-525A or piperonyl butoxide as a mixed function oxidase (MFO) inhibitor, isofenphos oxon lost all the inhibitory activity. On the other hand,N-isopropyl homologue partly retained its AChE activity and completely lost the activity by the addition of methimazole as a flavin-containing monooxygenase (FMO) inhibitor. Comparison of these data with insecticidal activity suggested the involvement of another bioactivation mechanism. Peracid oxidation as a model of biological oxidation resulted in the production of des-N-alkyl compound (HO-PO) in addition toN-desalkyl compound (H2N-PO), which suggested the structure of the activated form as an oxidized form of phosphoramide moiety of the starting compound. Three structures were considered from a computational chemistry point of view.N-Oxide structure (RNH(O)-PO) is isomerized toO-aminophosphate structure (RNHO-PO) depending on the alkyl substituent on the nitrogen atom, and the latter is the intermediate to give des-N-alkyl compound, which has no AChE inhibitory action. The formation ofO-aminophosphate structure from the adduct of the starting compound with peracid was analyzed using model compounds by the theory of Frontier orbital. Also,O-aminophosphate structure might be producedviaphosphorus oxyoxonate structure (RNH-P(OO)). The possibility of hydroxylamine structure (RN(OH)-PO) to giveN-oxide orO-aminophosphate structure was small if any in this model phosphoramidate compounds. In any way,O-aminophosphate structure is most probable as a novel activated form mainly formed by FMO or peracid.

Insecticidal properties of 3-aminopropyl(methyl)phosphinic acid and its effect on K +-evoked release of acetylcholine from cockroach synaptosomes

3-Aminopropyl(methyl)phosphinic acid (APMP), a potent agonist of mammalian GABA B receptors, caused prostration in houseflies ( Musca domestica L.) on injection into their thoraces, with an ED 50 value of 0.42 μg/fly. The 48-h LD 50 values of APMP were estimated to be 0.45 and 5.6 μg/fly in the presence and absence of piperonyl butoxide, a mixed-function oxidase inhibitor, respectively. Analogues of APMP, bearing a longer or shorter side chain by a CH 2 unit, or a phenyl group in the place of the methyl group, were without effects. In perfusion assays with synaptosomes prepared from the thoracic/abdominal nerve cords of cockroaches ( Periplaneta americana L.), 1 mM APMP reduced K +-evoked acetylcholine release to 10.4% of the control. These findings indicate that the physiologically important site of action of APMP, which might be implicated in neurotransmitter release, is present in insect neurons.

Association of cytochrome P450 induction with oxidative stress in vivo as evidenced by 3-hydroxylation of salicylate

1. Previous studies have shown that formation of 2,3-dihydroxybenzoate (2,3-DHB) from salicylate in vivo is a sensitive and specific marker of. OH radical generation, since 2,3-DHB is formed exclusively by. OH radicals, whereas both. OH radicals and cytochrome P450 (CYP) contribute to the production of 2,5-DHB. In the present study the salicylate-hydroxylation assay was used to examine whether CYP induction by the administration of dexamethasone, phenobarbital or β-naphthoflavone to the male rat led to oxidative stress in vivo. 2. Dexamethasone was used under conditions that induced an ~50-fold induction of CYP P4503A expression in liver microsomal protein. Treatment with dexamethasone caused a 17.2-fold increase in 2,3-DHB plasma concentration compared with control animals. An increase in total hydroxylated salicylate (2,3-DHB plus 2,5-DHB) of 133.5 μmol/l plasma was produced, of which - assuming that the attack by. OH in position 3 or 5 of salicylate occurs at a similar rate - 10.9 μmol/l were due to. OH radical attack and 122.6 μmol/l due to metabolism by CYP. 3. Phenobarbital led to a 4.7-fold increase in 2,3-DHB plasma concentration under conditions that induced CYP P4502B and 3A. An increase in total hydroxylated salicylate of 34.3 μmol/l plasma was observed, 2.0 μmol/l due to. OH radical attack and 32.3 μmol/l due to metabolism by cytochrome P450. 4. In contrast to dexamethasone and phenobarbital, β-naphthoflavone did not cause a significant increase in 2,3-DHB plasma concentrations. 5. SKF 525A, a mixed-function oxidase inhibitor, caused a significant reduction of mean 2,5-DHB plasma concentration by 35% (p < 0.001), whereas 2,3-DHB was not significantly reduced, indicating that in contrast to the situation after induction by dexamethasone or phenobarbital,. OH radical generation by constitutive CYP contributes only to a minor degree to total in vivo. OH radical generation. 6. This study shows for the first time, to the authors' knowledge, that induction of some (but not all) P450s is associated with the production of hydroxyl radicals in vivo.

Molluscicidal and Anti-AChE Activity of Tertiary Mixtures of Pesticides

We studied the toxicity and in vivo inhibition of acetylcholinesterase (AChE) by the organophosphate Nuvan (dichlorvos); Nuvan mixed with a mixed function oxidase inhibitor, piperonyl butoxide (PB); Nuvan with a pyrethroid Decis (deltamethrin); and a tertiary mixture of Nuvan, PB, and Decis in the snail Lymnaea acuminata. Nuvan was highly toxic to the snail and was a strong inhibitor of AChE activity. Both PB and Decis act synergistically with Nuvan when given together in a 1:5 and 1:46 ratio, respectively. When a tertiary mixture of Nuvan, Decis, and PB was given in a 1:46:5 ratio, the toxicity of the mixture was higher in comparision to the binary mixture of Nuvan + PB or Nuvan + Decis.

Effect of piperonyl butoxide or allopurinol on cyanide-induced lipid peroxidation in mouse brain

The effect of piperonyl butoxide or allopurinol on cyanide-induced lipid peroxidation was investigated using homogenates from whole brain of mice. The brain homogenate exposed to a low concentration of potassium cyanide (10, 50, or 100 μM) was significantly increased in their concentration of malondialdehyde (MDA)+4-hydroxyalkenals (4-MDA) as compared to control samples, in a concentration-dependent manner. The increased lipid peroxidation induced by cyanide was inhibited by piperonyl butoxide (1 mM), an inhibitor of mixed function oxidase, but not by allopurinol (0.1 mM), an inhibitor of xanthine oxidase. Furthermore, when a brain homogenate heated at 86°C for 1 min was incubated with or without cyanide at 37°C for 20 min, MDA+4-MDA levels in the homogenate were not changed between cyanide treatment and untreated. An intraperitoneal injection of piperonyl butoxide (1 g/kg) significantly inhibited cyanide-induced seizures in mice. These results suggest that cyanide-induced seizures may be partly involved in the lipid peroxidation produced by the heat unstable and piperonyl butoxide dependent factors in brain.

Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo.

S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol, gave rise to a 119-fold increase in ethanol-derived blood acetaldehyde (AcH) levels-a consequence of the inhibition of hepatic aldehyde dehydrogenase (A1DH)-when compared to control animals not receiving 4. The corresponding O-methylisourea was totally inactive under the same conditions, suggesting that differential metabolism may be a factor in this dramatic difference between the pharmacological effects of O-methylisourea and 4 in vivo. The S-n-butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10) was slightly less active. However, blood ethanol levels of all experimental groups were indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10 plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism. In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase and yeast A1DH, the requirement for microsomal activation for the inhibition of A1DH activity was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced in vivo inhibited A1DH directly, or spontaneously released cyanamide, an inhibitor of A1DH. Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea (11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of the liver and kidneys when administered to rats.

Mechanism of Resistance to Deltamethrin inCydia pomonella(L.) (Lepidoptera: Tortricidae)

The pathways for deltamethrin detoxification were studied in fifth instar larvae of two resistant strains ofCydia pomonellaobtained by mass selection (with deltamethrin (Rv) or diflubenzuron (Rt)) of resistant field populations from southeastern France. Compared to a laboratory susceptible strain (S), these strains exhibited respectively a 3170- and 1600-fold resistance ratio to topically applied deltamethrin. The esterase inhibitorS,S,S-tributyl phosphorotrithioate synergized deltamethrin in both resistant and susceptible strains, whereas the mixed-function oxidase (mfo) inhibitor piperonyl butoxide (PBO) synergized deltamethrin only in R strains (Rv and Rt). The two inhibitors together totally overcame the deltamethrin resistance of Rv and Rt strains. While ethoxycoumarin deethylase activities, measured at individual level in microtitration plates, were higher in R strains than with S, those of glutathioneS-transferases showed little difference and those of esterase showed no difference. Moreover, the involvement of mfo in the resistance to deltamethrin was confirmed byin vivometabolization of [14C] deltamethrin by guts of fifth instar larvae. Metabolites resulting from the ester bond hydrolysis, such as phenocyanol and PBald, were common to S and R strains. The mfo-dependent hydroxylated derivatives (torc-CHO-t-deltamethrin, 4′ or 5-HO-deltamethrin and 4′ or 5-HO-PB-acid) were generated only in Rv and Rt strains and were inhibited by PBO. These results may provide the basis for further studies in genetic of resistance inC. pomonella.

Effect of melatonin, piperonyl butoxide, or cobalt chloride on l-cysteine-induced lipid peroxidation in homogenate from whole brain of mice

Brain homogenates exposed to a low concentration of l-cysteine (0.05, 0.1, 0.5, 1.0, and 2.0 mM) were significantly increased in their concentration of malondialdehyde (MDA) + 4-hydroxyalkenals (4-HDA) as compared to control samples, in a concentration-dependent manner. The increased lipid peroxidation by l-cysteine was attenuated or completely abolished by co-incubation with melatonin (2 mM), a potent free radical scavenger, piperonyl butoxide (1 mM), an inhibitor of mixed function oxidase, or cobalt chloride (0.01, 0.02 or 0.05 mM), another inhibitor of mixed function oxidase, but not allopurinol (0.1 mM), an inhibitor of xanthine oxidase. In addition, when a brain homogenate heated at 85 °C for 1 min was incubated with or without l-cysteine at 37 °C for 20 min, MDA + 4-HDA levels in the homogenate was not changed between l-cysteine and control. These results suggest that l-cysteineinduced lipid peroxidation may be involved in the enzymatic mixed function oxidation systems in brain.