Metabolism of tetramethrin in houseflies in vivo

Houseflies susceptible to pyrethroids were found to metabolize in vivo radioactive tetramethrin (phthalthrin) or 3,4,5,6-tetrahydrophthalimidomethyl dl-trans chrysanthemumate mainly to N-(hydroxymethyl) 3,4,5,6-tetrahydrophthalimide and chrysanthemumic acid. Smaller amounts of oxidation products of tetramethrin or chrysanthemumic acid were formed. Pretreatment with piperonyl butoxide had little effect on the cleavage of the ester bond of tetramethrin, but another synergist NIA 16388 remarkably inhibited this reaction.     

A standardised in vivo and in vitro test method for evaluating tick repellents

The threat of transmission of Lyme borelliosis and tick-borne encephalitis by ixodid ticks has resulted in an increasing number of tick repellents coming onto the market. To allow proper evaluation of the efficacy of different types of compounds and their formulations, there is a need for standardised methods for testing ticks repellents. Ticks show a marked negative geotactic response following contact with a potential host, i.e., they climb up in order to locate attachment and feeding sites, whereas exposing ticks to repellents induces positive geotaxis, i.e., ticks walk downwards or drop off the treated host or substrate. We describe here complementary tests that employ these geotactic responses to evaluate repellents: one in vitro on a warm glass plate and the other on the lower human leg (shin). The compounds tested were DEET, EBAAP, icaridin, capric acid, lauric acid, geraniol, citriodiol, citronella essential oil and lavender essential oil, all non-proprietary ingredients of widely distributed tick repellent formulations.     

Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L.

The efficacy of the essential oil and various organic extracts from flowers of Cestrum nocturnum L. was evaluated for controlling the growth of some important phytopathogenic fungi. The oil (1000 ppm) and the organic extracts (1500 μg/disc) revealed antifungal effects against Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum in the growth inhibition range of 59.2-80.6% and 46.6-78.9%, respectively, and their MIC values were ranged from 62.5 to 500 and 125 to 1000 μg/mL. The essential oil had a remarkable effect on spore germination of all the plant pathogens with concentration and time-dependent kinetic inhibition of P. capsici. Further, the oil displayed remarkable in vivo antifungal effect up to 82.4-100% disease suppression efficacy on greenhouse-grown pepper plants. The results obtained from this study may contribute to the development of new antifungal agents to protect the crops from fungal diseases.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Inhibition of termite cellulases by carbohydrate-based cellulase inhibitors: Evidence from in vitro biochemistry and in vivo feeding studies

Efficacy of three prototype termite cellulase inhibitors, cellobioimidazole (CBI), fluoromethylcellobiose (FMCB) and fluoromethylglucose (FMG) was investigated using biochemical and feeding assays. Optimal conditions for measuring endoglucanase, exoglucanase and β-glucosidase activities were first determined. The three inhibitors were then tested under optimal conditions against enzyme fractions that represented endogenous (foregut/salivary gland/midgut) and symbiotic (hindgut) cellulases. In vitro, CBI and FMCB both inhibited exoglucanase and β-glucosidase activity (I₅₀s in nM and mM range, respectively). Feeding assays showed significant impacts on both survivorship and feeding stimulation by FMCB and CBI. Enzymatic measurements on feeding assay survivors showed impacts on all three cellulase activities by CBI and lesser impacts by FMG and FMCB. Validative bioassays with the sugars glucose, maltose and cellobiose showed no feeding stimulation or mortality as occurred in feeding inhibitor bioassays. These results indicate efficacy for two cellobiose-based inhibitors, FMCB and CBI, suggesting potential for these inhibitors as novel termite control agents.     

The apparent in vivo neuromuscular effects of the δ-endotoxin of Bacillus thuringiensis var. israelensis in mice and insects of four orders

Alkaline-dissolved crystal δ-endotoxin from Bacillus thuringiensis var. israelensis (serovar H 14) was injected into mice and seven species of insects representing the orders Lepidoptera, Orthoptera, Coleoptera, Hemiptera, and Diptera. High in vivo toxicity, at 1 to 5 ppm (μg toxin/g body wet wt), was observed with mice and some insects, including some that are not sensitive to the toxin when administered orally. Neuromuscular effects were observed when the toxin was injected directly into the body cavity of the test animals. Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.     

The metabolism of DDT in vivo by the Japanese quail (Coturnix coturnix japonica)

Male and female Japanese quail (Coturnix coturnix japonica) were given intraperitoneal injections of [¹⁴C]DDT in ethanol at a rate of 13.4 mg/kg body wt. Fifty-six days later the tissues and droppings were analysed for total ¹⁴C and metabolites. The rate of loss of ¹⁴C in droppings was very similar in males and females. The maximal rate was reached on the third day, and 65–66% of the injected dose was voided by the fifty-sixth day. Ninety-three to ninety-four percent of the ¹⁴C in droppings and 83–90% of the ¹⁴C in tissues were extracted by solvents. Combined extracts from males and females were used for determination of DDT and its metabolites. Expressing all results as percentages of injected dose, the following were isolated from droppings: DDA (24%), DDT (3%), DDD (5.1%), DDE (11%), and uncharacterised polar metabolites (17%). Twenty-five percent of the dose was retained in the tissues and this was largely accounted for as DDT (10.4%) and DDE (10.5%). Of the total metabolites found 31% was DDE (almost equally divided between tissues and droppings) and 35% was DDA (almost entirely in droppings). Since DDD was not found in significant quantities in tissues, the substantial quantities in droppings were probably produced from DDT by the action of microorganisms.     

Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin

Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68 ± 5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H₂O₂, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150 μg/l (DI: 239.62 ± 6.21) and 0.300 μg/l (270.63 ± 2.09) compared to control (150.25 ± 4.38) but no effect was observed at 0.075 μg/l (168.50 ± 10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H₂O₂), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.     

Effect of organophosphates in vivo upon acetylcholinesterase isozymes from housefly head and thorax

Four soluble acetylcholinesterase isozymes of head and three from the thorax of housefly (Musca domestica L.) were separated by polyacrylamide gel electrophoresis. Their inhibition in vivo was studied after poisoning with an LD₅₀ of four organophosphates: malaoxon, paraoxon, diazinon and dichlorvos. The isozymes differed greatly in their degree of inhibition. Thoracic isozymes were found to be more sensitive to inhibition than head isozymes. In surviving flies, the recovery rates of head isozymes were much faster than thoracic isozymes. In vitro studies showed that thoracic isozymes differed 4.1- and 2.9-fold in their K_m and V_max.     

棉铃虫C型凝集素抑制多角体病毒体内增殖

为探究C型凝集素（C-type lectin）在病毒传播中的可能作用,采用半定量RT-PCR法检测了棉铃虫Helicoverpa armigera不同C型凝集素在核型多角体病毒（nuclearpolyhedrosis virus,NPV）感染后的表达变化,并通过real-time qPCR和RNA干扰技术研究了NPV在棉铃虫体内的增殖状况及对棉铃虫致死率的影响。结果显示,在感染NPV后,棉铃虫血细胞C型凝集素Ha-lectin1和Ha-lectin5基因表达变化明显,其中Ha-lectin1在血细胞和脂肪体中的表达显著上调;注射dsRNA后,Ha-lectin1基因相对于actin的表达量均值由0.19下降到0.04,下降幅度达79%,显示Ha-lectin1基因被成功干扰。随着Ha-lectin1基因的沉默,病毒滴度由0.0015快速升高到1.95,棉铃虫的死亡率也随之增加到97.7%。表明C型凝集素能够抑制NPV在棉铃虫体内的增殖。     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Enhancement of dye-sensitized phototoxicity to house fly larvae in vivo by dietary ascorbate,diazabicyclooctane, and other additives

Several reagents reported to be singlet oxygen quenchers and/or radical scavengers and to be protectants against photooxidative damage to isolated systems in vitro were examined, in vivo, for protective effect on the larvae of the house fly (Musca domestica L.; Diptera:Muscidae). A standardized erythrosine-sensitized phototoxic test procedure was used. β-Carotene appeared to show some protective effect. Other dietary additives exhibited no measurable degree of protection to the larvae in vitro; on the contrary, mortality increased in the presence of butylated hydroxytoluene, ascorbate, and diazabicyclooctane.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Structure-activity relationships of DDT-type analogs based on in vivo toxicity to the sensory nerves of the cockroach, Periplaneta americana L

Forty-three DDT-type compounds were applied in saline suspension to the crural nerve of Periplaneta americana L. and the threshold concentration (ED₅₀) to produce trains of impulses was determined together with the frequency of appearance of repetitive afterdischarge. These quantitative neurological measures were evaluated in multiple regression analyses of structural parameters including van der Waal's volume, the F and R components of Hammet's σ, and the hydrophobic constant Π. This structure-activity analysis provides an accurate estimation of the intrinsic toxicity of the DDT analogs. The results affirm previous working theories that the bulk of the functional groups within the DDT framework is the primary factor relating to activity. However, conformation is also an important parameter.     

The in vitro metabolism of azinphosmethyl by mouse liver

The in vitro metabolism of methoxy-¹⁴C- or ³²P-azinphosmethyl by subcellular fractions from mouse liver was studied. The major degradative activity was associated with the microsomal and soluble fractions. Since the microsomal activity required NADPH and was inhibited by carbon monoxide, it is reasonable to assume that the mixed function oxidases were involved. The microsomal system catalyzed the dearylation reaction resulting in the formation of dimethyl phosphorothioic acid and dimethyl phosphoric acid. The system was also responsible for the oxidative desulfuration of azinphosmethyl resulting in the formation of azinphosmethyl oxygen analog.     

Persistence of some organophosphorus insecticides in hen-house litter

Thin-layer chromatography was used to assess semi-quantitively the persistence of six organophosphorus insecticides in hen-house litter. Malathion disappeared within 4 h of application. Carbofenthion was the most persistent. Four weeks after treatment the proportions of the applied doses extracted from the litter were: carbofenthion 46%, phorate 21%, diazinon 13%, coumaphos 7% and fenitrothion 3%.     

Control of wireworms with organophosphorus and carbamate insecticides

Carbaryl, chlorfenvinphos, diazinon, disulfoton, parathion, phorate, fenitrothion, thionazin and trichlorphon were tested for their effectiveness in killing wireworms by broadcast treatments. Each insecticide was tested once or more in two field trials cropped with wheat and two cropped with potatoes. Phorate and parathion were very lethal to wireworms at 4 Ib active ingredient per acre (4–48 kg/ha). Thionazin and diazinon were intermediate in effectiveness and the other insecticides ineffective.     

Synergism of organophosphorus insecticides by diethyl maleate and related compounds in house flies

The toxicity of several organophosphorus and one carbamate insecticide for house flies is enhanced by simultaneous administration of diethyl maleate. Synergism factors vary from 2 to 116 and are strongly dependent on the combination of strain and insecticide studied. In general, it seems that thiono compounds are less synergized than their oxon analogs. Comparison of some analogs of maleic acid esters showed that the diethyl ester was not the most active compound. trans-Phenylbutenone was found to be a good alternative for diethyl maleate. Both diethyl maleate and trans-phenylbutenone deplete glutathione in the flies, but this depletion per se is certainly not the most important mode of action. In vitro experiments made clear that sufficient glutathione remained to permit a substantial insecticide degradation rate. Moreover, treatment of the flies with diethyl maleate 2–4 hr before the insecticide application resulted in a partial or complete disappearance of synergism in a period in which the glutathione concentration in the flies was still very low. Apart from this effect on the glutathione levels in the insect tissues it appeared from further experimental work in vitro that both compounds had a direct inhibitory effect on glutathione-dependent transferase(s) as well as on oxidative enzymes involved in insecticide degradation. The contribution of these reactions to the eventual synergism factor is discussed.     

The effect of O,S,S,-trimethyl phosphorodithioate on the in vivo metabolism of phenthoate in the rat

The in vivo metabolism of phenthoate (O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate) was followed in rats after oral administration of a nontoxic dose of 100 mg/kg. The same metabolic study was conducted following coadministration of 0.5% O,S,S-trimethyl phosphorodithioate (OSS-Me). When administered alone, phenthoate was metabolized principally by carboethoxy ester hydrolysis and cleavage of the PO and CS bonds, resulting in at least six metabolites. The primary urinary metabolite excreted was phenthoate acid. Coadministration of 0.5% OSS-Me did not alter the types of metabolites excreted. However, a reduction of the carboxylesterase-catalyzed product (phenthoate acid) was observed, indicating that the enzyme responsible for the major pathway of phenthoate detoxication was inhibited. Alternate detoxication processes did not compensate for the reduction in carboxylesterase-catalyzed detoxication. It was concluded that inhibition of the carboxylesterase enzymes is the major cause of the potentiation of phenthoate toxicity by OSS-Me.     

Metabolism of tetramethrin in houseflies and rats in vitro

Degradation of radiolabeled tetramethrin or 3,4,5,6-tetrahydrophthalimidomethyl dl-trans chrysanthemumate was tested in vitro by using abdomens of SK, lab-em-7-em, R_HOKOTA and P_y strains of houseflies and rat liver. The effect of NADH₂ and NADPH₂ on the metabolism of tetramethrin by housefly abdomen homogenate was slight, but phosphorothioates, their oxygen analogs, carbamate insecticides, NIA 16388, p-chloromercuribenzoic acid, and mercuric chloride showed marked inhibition. The enzyme activity was localized mainly in the microsomal fraction, where the major metabolites were 3,4,5,6-tetrahydrophthalimide (TPI) (a nonenzymatic reaction from N-(hydroxymethyl) 3,4,5,6-tetrahydrophthalimide, MTI) and chrysanthemumic acid. Smaller amounts of oxidized tetramethrins and chrysanthemumic acid were also produced. The cleavage of tetramethrin into MTI and chrysanthemumic acid was inhibited by such compounds as paraoxon, carbaryl, PCMB, NIA16388, and mercuric chloride. NADPH₂ or NADPH₂ plus carbon monoxide produced little effect. Similar results were obtained with rat liver microsomal fraction. It is presumed from the above findings that the cleavage is catalyzed either by a carboxyesterase or a hydrolase, and that some pyrethroids are metabolized in insects primarily through hydrolytic pathways. Metabolites from oxidative pathways (as in mammals) are formed in minor quantities.