Simultaneous determination of pyrethroid and pyrethrin metabolites in human urine by gas chromatography-high resolution mass spectrometry

A new developed gas chromatographic-high resolution mass spectrometric method for the sensitive simultaneous determination of trans-chrysanthemumdicarboxylic acid, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine is presented. These metabolites are biomarkers for an exposure to pyrethrum, allethrin, resmethrin, phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin or permethrin. Therefore, with the help of this method for the first time a complete assessment of exposure to pyrethroid and pyrethrin insecticides is possible. After acid hydrolysis and extraction with tert-butyl-methyl-ether the residue is derivatized with 1,1,1,3,3,3-hexafluoroisopropanol and analyzed by GC/HRMS in electron impact mode (detection limits < 0.1 microg/l) as well as in negative chemical ionization mode (detection limit < 0.05 microg/l urine).     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Bioassay for detecting active site insensitivity in horn fly (Diptera: Muscidae) larvae

A bioassay was used to detect active site insensitivity (knock-down resistance [kdr]) in pyrethroid resistant larvae of the horn fly, Haematobia irritans (L.). The larvae of the resistant population had KD50's 42.0-, 28.1- and 29.2-fold greater to permethrin, fenvalerate and lambda-cyhalothrin, respectively, compared with the susceptible population. In filter paper bioassays, resistant adult horn flies were 17 to 39.1 times less susceptible to the pyrethroids than susceptible adults at LC50. These results further document active site insensitivity as the major mechanism of pyrethroid resistance in the horn fly.     

Chronology of permethrin resistance in a southern Illinois population of the horn fly (Diptera: Muscidae) during and after selection by pyrethroid use

From 1985 through 1988, horn flies (Haematobia irritans (L)) collected at the Dixon Springs Agricultural Center (DSAC) in southern Illinois were tested in 22 h bioassays for permethrin resistance with residues on cotton cloths. The LC90 for a susceptible field population collected in June 1985 was 0.19 micrograms/cm2. In comparison, flies collected from pyrethroid-tagged cattle in 1985 and 1986 exhibited 25- to 116-fold resistance to permethrin. A 25-fold level of resistance allowed survival on treated cattle 8 wk after pyrethroid tag application. Flies representing the local background population were collected periodically from an untreated herd 2.4 km from the nearest cattle treated with a pyrethroid; these flies exhibited up to 18-fold resistance. Although pyrethroids were not used on DSAC animals after October 1986, all bioassays done in 1987 and 1988 indicated resistance levels of greater than or equal to 7-fold. The 95% confidence intervals for LC90s from all 1987 bioassays overlapped the confidence interval from the corresponding July 1986 estimate for resistant flies collected from pyrethroid-tagged cattle. Although some decline in resistance was evident in 1988, bioassays done at the end of the season produced resistance ratios of 7.4 and 15.3. Survivorship at a diagnostic dose indicated that resistance frequencies remained at 4-8% throughout 1988. Two years' abstinence from pyrethroid use was insufficient to allow an adequate decline in resistance levels.     

Influence of five pyrethroid insecticides on microbial populations and activities in soil

Laboratory tests were conducted to determine the effects of five pyrethroid insecticides—permethrin (FMC 33297) [3-phenoxybenzyl (±)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate]; FMC 45498 [(S)--cyano-3-phenoxybenzyl-(R)-cis-2-(2,2-dibromovinyl)-3,3-dimethylcyclopropanecarboxylate]; Shell WL 41706 [(±)--cyano-3-phenoxybenzyl 2,2,3,3-tetramethylcyclopropane-carboxylate]; Shell WL 43467 [(±)--cyano-3-phenoxy benzyl (±)-cis,trans-2-(2,2-dichlorovinyl)-3,3-dimethylcyclopropanecarboxylate]; and Shell WL 43775 [(±)--cyano-3-phenoxybenzyl (±)-2-(4-chlorophenyl)-3-methylbutyrate]—at 0.5 and 5g/g on microbial populations and activities in a sandy loam. The insecticides had antimicrobial activity in early stages of incubation. The populations recovered after 2 to 4 weeks and stimulatory effects on populations were also observed in later stages. No inhibition of acetylene (C₂H₂) reduction was evident with any of the insecticides. However, WL 43467 at both concentrations and permethrin and WL 41706 at 5 g/g increased nitrification after 4 weeks. Soil microbial respiration, as indicated by oxygen consumption, increased with increasing concentration of insecticides, suggesting the possibility of microbial degradation of the insecticides. Dehydrogenase activity showed that none of the insecticides inhibited formazan (2,3,5-triphenyltetrazolium formazan) formation, whereas urease activity was stimulated in most cases. The studies indicated that some of the pyrethroid insecticides may exert transient effects on populations and activities of the microflora in a sandy loam, but these were short-lived and minor in nature.     

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅱ:氧化代谢

氧化代谢的增强是引起家蝇对二氯苯醚菊酯产生抗性的因素之一.抗性家蝇多功能氧化酶的萘羟化活性、对二氯苯醚菊酯的氧化代谢能力和微粒体细胞色素P₄₅₀含量分别是正常家蝇的2、1.48、1.33倍.正常家蝇和抗性家蝇细胞色素P₄₅₀在对增效磷(SV₁)和氧化胡椒基丁醚(Pb)的敏感性上也存在着差异.SV₁与多功能氧化酶专一性抑制剂Pb一样,对该酶系催化的萘羟化活性及二氯苯醚菊酯的氧化代谢有明显的抑制作用,这种抑制作用是SV₁在家蝇体内对二氯苯醚菊酯增效的机理之一.SV₁对氧化代谢的抑制与它和微粒体细胞色素P₄₅₀相互作用形成非活性复合体有关.     

Effect of fluorine-containing ethers of permetrinic acid on inactivation kinetics of sodium influx into neuroblastoma cells (NEURO-2(alpha)]

A synthesis of a number of esters of (+/-)-cis, trans-3-(2,2-dichlorovinyl) -2,2-dimethylcyclopropanecarboxylic acid with polyfluorinated alcohols was carried out. Their effect on kinetics of inactivation of sodium current in neuroblastoma (Neuro-2a) cells by the patch-clamp technique in the whole-cell configuration was investigated.     

Evaluation of horn flies (Diptera: Muscidae) from a pyrethroid susceptible colony for general and permethrin esterase activities

In this study we describe a nonradioactive single-fly microassay for permethrin hydrolysis. We used this assay with a microplate assay for general esterase activity to evaluate the permethrin hydrolyzing and general esterase activities of aging pyrethroid-susceptible male and female horn flies, Haematobia irritans (L.). We found substantial gender- and age-related differences regarding general esterase activity, permethrin sensitivity, and permethrin hydrolyzing activity within the colony. Extracts of female flies collected 48 h after receiving their first blood meal yielded significantly greater esterase activity than male extracts. Aging female flies were more tolerant of permethrin than were male flies. In addition, a positive correlation was found to exist between the general esterase activity of aging females and their ability to hydrolyze permethrin.     

Susceptibility of insecticide-susceptible and wild house flies (Diptera: Muscidae) to abamectin on whitewashed and unpainted wood

Unpainted plywood panels treated with 0.1% abamectin (avermectin B1) provided greater than 90% control of house flies, Musca domestica L., susceptible to insecticides for 4 wk and greater than 70% control for 7 wk compared with 46-92% control observed with permethrin at the same time and rate of application. Efficacy of abamectin on whitewashed panels was similar to that observed on unpainted panels, whereas permethrin was ineffective on whitewashed panels at all rates tested (range, 0.001-0.1%) at all intervals after treatment. Bioassays of newly colonized house flies resistant to permethrin indicated that wild populations may be cross-resistant to abamectin.     

Release of piperonyl butoxide and permethrin from synergized ear tags on cattle and effects on horn fly mortality

A study was conducted to determine the release rates of piperonyl butoxide (PBO) and permethrin from synergized insecticidal cattle ear tags and their effects on mortality of the horn fly, Hematobia irritans irritans (L.) (Diptera: Muscidae). PBO was released from the ear tags at a higher rate than permethrin in both winter and summer trials. The cumulative release of PBO and permethrin from the ear tags at the end of 18 wk in the winter trial was 50.4 and 30.3%, respectively. The cumulative release of PBO and permethrin from the ear tags at the end of 18 wk in the summer trial was 66.7 and 44.7%, respectively. There was a significant correlation between the cumulative daily high ambient temperature (degrees C) and the cumulative release of both PBO and permethrin. Compared with the susceptible horn fly strain, the permethrin-resistant strain demonstrated 7.9- and 12.8-fold resistance to permethrin at the levels of LC50 and LC90, respectively. When exposed to filter paper wipes taken from the shoulders of cattle treated with the PBO-synergized permethrin tags from the summer trial, the resistant strain demonstrated reduced mortality compared with the susceptible strain. The mortality of the resistant strain at 2- and 3-h exposure exhibited a pattern of declining fly mortalities as a result of the decreased release of PBO and permethrin, as well as the decline in the ratio of PBO:permethrin released from the tags after 8 wk. A similar decline in horn fly mortalities was observed in the susceptible strain at 30-min exposure time that coincided with the pattern of reduced release of PBO and permethrin from the ear tags over the course of summer trial.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Effect of temperature on toxicity of three pyrethroids to horn flies

Predictive models describing best-fit regression equations for per cent mortality of horn flies as a function of temperature were determined for each of three pyrethroid insecticides (fenvalerate, flucythrinate and permethrin) over the temperature range 20-35 degrees C. Susceptible horn flies, Haematobia irritans (L.), were exposed to c. an LC70 dose of each pyrethroid using a residue-on-glass method. This technique used confined exposure in chambers with temperatures of 20, 25, 30 and 35 degrees C. Within this range, mortality was greatest at 25 degrees C with all three insecticides. Estimated temperature-mortality equations for each pyrethroid revealed different responses of horn flies to each of these insecticides. Horn flies exposed to flucythrinate demonstrated a linear mortality response that varied inversely with temperature. The response to permethrin was described by a quadratic equation, while the response to fenvalerate was best fitted by a cubic equation.     

Isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men

Human lipid intake contains various amounts of trans fatty acids. Refined vegetable and frying oils, rich in linoleic acid and/or alpha-linolenic acid, are the main dietary sources of trans-18:2 and trans-18:3 fatty acids. The aim of the present study was to compare the oxidation of linoleic acid, alpha-linolenic acid, and their major trans isomers in human volunteers. For that purpose, TG, each containing two molecules of [1-(13)C]linoleic acid, alpha-[1-(13)C]linolenic acid, [1-(13)C]-9cis,12trans-18:2, or [1-(13)C]-9cis,12cis,15trans-18:3, were synthesized. Eight healthy young men ingested labeled TG mixed with 30 g of olive oil. Total CO(2) production and (13)CO(2) excretion were determined over 48 h. The pattern of oxidation was similar for the four fatty acids, with a peak at 8 h and a return to baseline at 24 h. Cumulative oxidation over 8 h of linoleic acid, 9cis,12trans-18:2, alpha-linolenic acid, and 9cis,12cis,15trans-18:3 were, respectively, 14.0 +/- 4.1%, 24.7 +/- 6.7%, 23.6 +/- 3.3%, and 23.4 +/- 3.7% of the oral load, showing that isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men.     

Horn fly (Diptera: Muscidae) insecticide resistance in Kentucky and Arkansas

The effect of previous insecticide use patterns for horn fly control on the susceptibility spectrum of horn fly (Haematobia irritans [L.]) populations from Kentucky and Arkansas is described. Populations of horn flies from both states were tested with three pyrethroids (cyhalothrin, cypermethrin, and permethrin), three organophosphates (diazinon, pirimiphos methyl, and tetrachlorvinphos), and a chlorinated hydrocarbon (methoxychlor). Dose-mortality data indicated insecticide resistance in Arkansas and Kentucky. Two permethrin-resistant horn fly populations in Kentucky that did not have a history of exposure to methoxychlor were cross-resistant to this chlorinated hydrocarbon. Horn fly populations from both states with a history of at least three consecutive years of exposure to various pyrethroid ear tags were subsequently exposed to cattle tagged with cyhalothrin-impregnated ear tags for 15-16 wk. Such exposure resulted in a decrease in susceptibility to this pyrethroid (ranging from approximately 30 to greater than 100-fold) when compared with levels before treatment. Horn fly populations from Arkansas resistant to cyhalothrin (as a result of exposure to cyhalothrin ear tags) were cross-resistant to pirimiphos methyl. Seasonal exposure of an Arkansas and Kentucky horn fly population to cattle with ear tags impregnated with pirimiphos methyl resulted in a significant decrease in susceptibility to this organophosphate.     

High levels of pyrethroid resistance in horn flies (Diptera: Muscidae) selected with cyhalothrin.

Lambdacyhalothrin cattle ear tags controlled horn fly, Haematobia irritans (L.), for 14 wks or longer during 1986-1988 in Georgia, USA. In 1989 and 1990, control of < 50 horn flies per side of cow was achieved for < or = 4 wk because of high levels of pyrethroid resistance in horn flies selected with lambdacyhalothrin. The highest resistance ratios (RRs) were seen in 1989. These were 498 for lambdacyhalothrin; 92,000 for fenvalerate; and 54 for permethrin. RRs for cypermethrin as high as 8,800 were estimated in 1990 when the RR for fenvalerate was only 1,060. No cross-resistance to diazinon was detected. These high levels of pyrethroid resistance seem to have a large component of metabolic resistance. Synergistic coefficients as high as 3,600 were determined by addition of nonlethal amounts of piperonyl butoxide. Resistance development in a no-pyrethroid-use area indicates movements of > or = 3km by sufficient numbers of horn flies can significantly change the RR.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅰ:水解代谢

水解代谢在家蝇对二氯苯醚菊酯抗性中起重要作用。正常家蝇和抗性家蝇酯酶在对SV₁、SV₂等抑制剂的敏感性和电泳性质上存在着差异。抗性家蝇酯酶水解二氯苯醚菊酯的活性较高,水解乙酸-α-萘酯的活性相对比正常家蝇要低。SV₁及其在体内的代谢产物SV₂在离体和活体情况下对家蝇酯酶都有明显的抑制作用。SV₁和SV₂抑制相同的酯酶电泳条带,但SV₁的抑制作用相对小一些。SV_t对酯酶的抑制是它在家蝇体内对二氯苯醚菊酯增效的机理之一。     

Residual activity of microencapsulated permethrin against stable flies on lactating dairy cows

Emulsifiable concentrate and microencapsulated formulations of permethrin were evaluated for residual activity against stable flies on lactating dairy cows. Cows were treated in the field with each formulation and hair was clipped from the leg and shoulder area, and bioassayed at 1, 2, 3, 4, 7 and 14 days after treatment. Significantly more stable flies died when exposed to hair sampled 3, 4 and 7 days after treatment from the shoulder than from the lower leg. Analysis with gas chromatography of hair samples showed no detectable permethrin residues on shoulder or leg hair 72 h after treatment with the emulsifiable concentrate formulation. Microencapsulated permethrin was still detectable on hair sampled from both locations 7 days after treatment. The permethrin concentration on the leg hair was approximately 50% of the shoulder hair concentration after 3 days, with the leg hair residue dropping to 31% of shoulder level after 7 days.     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).