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An Enzyme-Linked Immunosorbent Assay for the Detection of Esfenvalerate Metabolites in Human Urine
Abstract
The pyrethroids are one of the most heavily used insecticide classes in the world. Sensitive and rapid analytical techniques are needed for assessments of human exposure to these compounds. Highly sensitive and selective ELISAs for glycine conjugates of esfenvalerate key metabolites phenoxybenzoic acid (PBA) and s-fenvalerate acid (sFA) were developed. Rabbits were immunized with either N-(3-phenoxybenzoyl)-4-amino-L-phenylalanine-fetuin or N-[(S)-4-chloro-2-(methylethyl)benzeneacetyl]-4-amino-L-phenyla lan ine -fetuin, and all sera were screened against numerous coating antigens. The antibodies with the least interference and best sensitivity were optimized and characterized. The I(50)s for sFA-glycine and PBA-glycine in buffer were found to be 0.40 +/- 0.12 microg/L (1.47 +/- 0.44 nmol/L) and 0.42 +/- 0.18 microg/L (1.56 +/- 0.67 nmol/L), respectively. Both assays exhibited high selectivity. Little or no cross reactivity to the parent compound and other metabolites was measured. The matrix effects of urine were investigated. Solid-phase extraction (SPE) strategies were used in an attempt to decrease the matrix effects and increase the sensitivity of the overall method. The limit of quantitation (LOQ) for both sFA-glycine and PBA-glycine in urine with SPE is 1.0 microg/L (3.70 nmol/L). These assays could be used as markers of exposure for monitoring biological samples.
... Because major pyrethroids such as permethrin, cypermethrin, and cyfluthrin contain the PB 1 group and PBA is a common metabolite form or intermediate, PBA conjugates might be suitable biomarkers of human exposure. In the previous study, we developed a sensitive and selective immunoassay for detecting one of the possible pyrethroid metabolites, a PBA-glycine conjugate (14). However, no study has specifically determined the nature of the conjugates of pyrethroid metabolites in humans. ...
... Immunization. The production of Ab was made following the protocol reported earlier (14). Briefly, two New Zealand white rabbits were immunized (rabbit nos. ...
... In Dr. Leng's lab, these urine samples have been analyzed for the total FPBA after hydrolysis via GC-MS. To test which form of conjugate is predominant in the urine, these samples were tested by different immunoassays including the PBA-glycine ELISA (14). Each urine sample was split into three aliquots. ...
The pyrethroids are one of the most heavily used insecticide classes in the world. It is important to develop sensitive and rapid analytical techniques for environmental monitoring and assessment of human exposure to these compounds. Because major pyrethroids contain a phenoxybenzyl group and phenoxybenzoic acid (PBA) is a common metabolite form or intermediate, PBA might be used as a biomarker of human exposure to pyrethroids. A sensitive and selective immunoassay for the common pyrethroid metabolite PBA was developed. Rabbits were immunized with 3-[4-(3-carboxyphenoxy)phenoxy] N-thyroglobulin ethylamine. All sera were screened against numerous coating antigens. The assay with the least interference and the best sensitivity was optimized and characterized. The average IC 50 for free PBA was 1.65 ng/mL. No cross-reactivity was measured to parent pyrethroids and other metabolites. Urine matrix effects can be eliminated by simple dilution. Results from urine samples from exposed workers suggest that this PBA immunoassay might be suitable as a monitoring tool for human exposure to pyrethroids.
... Another problem arises from matrix interference. Fruit, vegetable, and other food extracts, as well as soil samples, contain organic and inorganic substances that may interfere directly or indirectly with the assay, increasing variability and decreasing efficiency and monitoring capability (19,29,39). Such matrix interferences may affect the Ab-analyte binding, increase nonspecific binding of reagents, inactivate the Ab or reporting enzyme, or introduce a high background because of sample pigmentation or autofluorescence. ...
... A large variety of food, environmental, and biological samples has been tested for pyrethroids or pyrethroid metabolites. Among those are various water samples (river and lake water, industrial tap water, and agricultural runoff) (19,22,24,25,28,32,52), urine (18,27,29), milk (23), tea (25), wine, fruits and vegetables (20,25), whole or ground wheat or barley grains (32,38,39), and soil (25,32). Several approaches were used in these studies to cope with the problem of matrix interference with ELISA detection. ...
... Several approaches were used in these studies to cope with the problem of matrix interference with ELISA detection. The most common approaches being sample dilution with an appropriate buffer (18,20,27,29,32) or SPE using C 18 (19,24,28,52), C 8 (52), or alumina columns (39) or multistep extraction procedures with organic solvents had to be employed (23). ...
Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.
... These methods are relatively intensive, timeconsuming, and expensive, and not particularly suitable for large numbers of samples. An immunoassay would provide a sensitive, selective, and rapid method for the detection of this pyrethroid at trace levels (10)(11)(12)(13)(14)(15)(16). ...
... Hapten Synthesis and Verification. Syntheses of the haptens were carried out as outlined in Figure 2. All reactions were straightforward and followed procedures employed in preceding publications (12)(13)(14)23). NMR spectral data supported all structures, and mass spectra further confirmed the structures of the final haptens. ...
A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate and 3-[(+/-)-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropan ecarbonyloxy]methyl]phenoxyacetic acid, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized. The I(50) for deltamethrin was 17.5 +/- 3.6 microg/L, and the lower detection limit was 1.1 +/- 0.5 microg/L. This ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin, phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin were analyzed according to this method. Good recoveries and correlation with spike levels were observed.
... Sensitive enzyme-linked immunosorbent assays (ELISAs) have been developed for the pyrethroid metabolite 3-PBA [33] and its glycine conjugate [29]. ...
... Since the cis-/trans-DCCA metabolite is a common structural group in some pyrethroids, including permethrin, cypermethrin, and cyfluthrin, the assay is class-specific for the metabolism study of pyrethroids containing DCCA. This assay may be a useful and significant tool together with the immunoassays for a PBA-glycine conjugate [29] and free PBA metabolite [33] as biomarkers of exposure to pyrethroids, including permethrin. ...
Permethrin is the most popular synthetic pyrethroid insecticide used in agriculture and public health. For the assessment of human exposure to permethrin, a competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the glycine conjugate of a major metabolite, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was developed based on a polyclonal antibody. An assay based on an antibody with a high sensitivity was optimized and characterized. The IC50 value and the detection range for trans-DCCA-glycine, in the assay buffer were 1.2 and 0.2-7.0 microg/L, respectively. The antibody recognized trans-DCCA-glycine and the mixture of cis-/trans-DCCA-glycine with an isomer range from 30:70 to 50:50 nearly equally. Little or no cross-reactivity to permethrin and its other free metabolites or glycine conjugates was measured. The integration of the ELISA and solid-phase extraction which was used to reduce the matrix effect from human urine samples provided for analysis of total cis-/trans-DCCA-glycine at low parts per billion levels in the samples. The limit of quantitation of the target analyte was 1.0 microg/L in urine with a limit of detection of 0.1 microg/L in buffer. This assay might be a useful tool for monitoring human exposure to permethrin.
... Nowadays, α-cyano pyrethroids are being extensively used as insecticides in domestic, agriculture, horticulture, public health, and veterinary applications, mainly due to its low mammalian toxicity and its activity against a broad range of pests [1][2][3]. Cyphenothrin, λ-cyhalothrin, cypermethrin, and deltamethrin (see Fig. 1) among other synthetic pyrethroids have reported improved physicochemical properties and biological actions compared to their natural analogues. Among them, deltamethrin have been presented as one of the more lipophilic and stable insecticide-permethrins, and in consequence, its residues have been detected in agricultural products [4], food [5,6], and surface waters [7,8]. ...
The study of an enzyme-linked immunosorbent assay (ELISA) and an amperometric biosensor for the detection of the pyrethroid deltamethrin in seawater is reported. The preparation of specific polyclonal antibodies is addressed using two immunizing haptens based on deltamethrin and cypermethrin compounds, with a spacer arm placed at the cyano residue in the pyrethroid structure. Different conjugates based on bovine serum albumin and aminodextran are prepared depending on the lipophilic profile of the competitor haptens studied. A reproducible and sensitive indirect competitive ELISA is developed, reaching a limit of detection of 1.2 ± 0.04 μg L−1 and an IC50 value of 21.4 ± 0.3 μg L−1 (both n = 3). For validation of the assays described, artificial seawater samples fortified with deltamethrin are analyzed. For the ELISA assay, these accuracy studies reported a slope of 0.904. An amperometric immunosensor is developed using the same immunoreagents and achieving a comparable detectability in terms of LOD of 4.7 μg L−1, measuring seawater without any pretreatment. These results suggest that both techniques can be used as rapid and simple analytical methods for deltamethrin quantification in seawater samples, which are great candidates for initial environmental screening programs.
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... The optimal concentrations of phage and antibody were determined by the checkerboard titration method for the competitive and noncompetitive phage ELISAs [8,30]. The experimental parameters, including the ionic strength, pH value and organic solvent, were sequentially studied to determine the optimal conditions to achieve the maximum sensitivity. ...
... 94 Recently, several immunoassays have been developed to assess human exposure to alachlor, 95,96 atrazine, 97,98 metolachlor, 99 and pyrethroids. 100 In the case of the herbicide atrazine, the mercapturic acid conjugate excreted in human urine 101 is a specific biomarker for exposure. A sensitive immunoassay has been developed for this metabolite 97 that can be detected at 0.1 µg L −1 in urine. ...
... The optimal concentrations of phage-displayed peptide and antibody were confirmed using the two-dimensional checkerboard method [24]. Selection of the phage-displayed peptide was performed by phage ELISA. ...
... The optimal concentrations of coating antigen and antibody were conrmed using the two-dimensional checkerboard method. 25 The experimental parameters including the organic solvent, ionic strength, and buffer pH value were sequentially studied to improve the sensitivity of the immunoassay. Competitive curves were run in PBS solutions containing different concentrations of methanol (from 5 to 30%, v/v) and Na + (0.1, 0.2, 0.3, 0.4 and 0.5 mol L À1 ) as well as with various pH values (5.5, 6.5, 7.5 and 8.5), and the evaluations of the parameters were based on the A max /IC 50 , IC 50 and the squared coefficients of correlation (R 2 ) of the linear equation. ...
An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on monoclonal antibodies (MAbs) for benzothiostrobin has been developed. The hapten of benzothiostrobin was synthesized and conjugated to bovine serum albumin and ovalbumin to generate an immunogen and a coating antigen. The immunogen was used to immunize BALB/c mice, resulting in anti-benzothiostrobin MAb. Under optimal conditions, the half maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed ic-ELISA were 7.55 and 0.428 μg L−1, respectively. The cross-reactivity (CR) was less than 0.05% for the tested structural analogues and regarded as negligible except for pyraclostrobin, which exhibited a CR of 0.34%. The recoveries of benzothiostrobin ranged from 80.43 to 113.83% in environmental and agricultural samples, respectively, which conformed to the requirements for residue detection. The amount of benzothiostrobin detected by ic-ELISA in the samples was significantly (R2 = 0.9894, y = 1.0867x + 0.0318) correlated with that detected by high-performance liquid chromatography (HPLC). The current study indicates that the established ic-ELISA is a potentially useful tool for detecting benzothiostrobin in environmental and agricultural samples.
... A checkerboard titration assay was carried out with different amounts of the coating antigen (hapten-OVA) in CBS and of the antibody in PBS [21]. After the screening of the antibody and the coating antigens, an indirect competitive ELISA was developed as follows: the microplates were coated for 2 h at 37uC with 100 mL/ well of the coating antigen (0.3 mg L 21 ) in CBS. ...
In the European Union (EU), the use of diniconazole-M is no longer authorized. However, residues of diniconazole-M occur in various plant commodities.
A selective and simple analytical method for the trace level determination of diniconazole in soil, fruit, vegetables and water samples was developed based on immunoaffinity extraction followed by Enzyme-linked immunosorbent assay (ELISA) and the high-performance liquid chromatography (HPLC) analysis. The ELISA was based on monoclonal antibodies highly specific to diniconazole and was a fast, cost-effective, and selective screening method for the detection of diniconazole. The results of the ELISA correlated well with gas chromatography (GC) results, with the correlation coefficient of 0.9879 (n = 19). A simple gel permeation chromato- graphy clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The immunoaffinity column (IAC) capacity was 0.180 mg g(-1). The columns could be re-used approximately 20 times with no significant alteration in capacity. The recoveries from complex samples were in the range of 89.2% to 96.1% with a relative standard deviation (RSD) of 0.770%-6.11% by ELISA. The results were in good agreement with those obtained by HPLC method.
The IAC extraction procedure coupled with HPLC and ELISA analysis could be also used as alternative effective analytical methods for the determination of diniconazole concentrations in complex samples.
... Hapten V (3-phenoxybenzoic acid; Table 1), and 3-phenoxybenzyl alcohol were purchased from Aldrich (St. Louis, MO) ( Fig. 1). Coating antigen haptens IV, VI, VII, and VIII (Table 1) were prepared in the laboratory [20]. Methanol (GC Resolve grade) was obtained from Fisher Scientific (Pittsburgh, PA), and dimethyl sulfoxide (DMSO; 99.8%) was purchased from Aldrich (St. Louis, MO). ...
A general and broad class selective enzyme-linked immunosorbent assay was developed for the type II pyrethroid insecticides, such as cypermethrin, deltamethrin, cyhalothrin, cyfluthrin, fenvalerate, esfenvalerate and fluvalinate. Polyclonal antibodies were generated by immunizing with a type II pyrethroid immunogen ((RS)-α-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-carboxyl-cyclopropanecarboxylate) conjugated with thyroglobulin. Antisera were screened against nine different coating antigens. The antibody–antigen combination with the most selectivity for type II pyrethroids such as cypermethrin was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC50s of the optimized immunoassay were 78 μg l−1 for cypermethrin, 205 μg l−1 for cyfluthrin, 120 μg l−1 for cyhalothrin, 13 μg l−1 for deltamethrin, 6 μg l−1 for esfenvalerate, 8 μg l−1 for fenvalerate and 123 μg l−1 for fluvalinate. No cross-reactivity was measured for the type I pyrethroids such as permethrin, bifenthrin, phenothrin, resmethrin and bioresmethrin. This assay can be used in monitoring studies to distinguish between type I and II pyrethroids.
... The method used was similar to that previously described by Shan et al. (7) Microplates were coated overnight at 48C with 100 mL=well of the appropriate coating antigen concentration in 0.1 M carbonate-bicarbonate buffer (pH 9.6). After the coated plates were washed with PBST (PBS plus Tween-20: 8 g L À1 NaCl, 1.15 g L À1 Na 2 HPO 4 , 0.2 g L À1 KH 2 PO 4 , 0.2 g L À1 KCl, and 0.05% Tween, v=v), 200 mL of blocking solution (5% degreased milk in PBST) was added and incubated for 2 h at 378C. ...
Using semisolid methylcellulose HAT medium selection and non-competitive and competitive indirect enzyme-linked immunosorbent assay, three hybridomas that secreted antibodies for aflatoxin G(1) were selected after fusion of mouse SP2/0 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with the conjugate of aflatoxin G(1) (AFG(1)) and bovine serum albumin. The aflatoxin-modified protein used to immunize mice was produced chemically by activating aflatoxin G(1) to a 9,10-epoxide derivative, which then covalently bound to the protein. All of these antibodies were found to be specific to AFG(1), and reacted strongly with aflatoxin G(2). These antibodies were designated 1C8, 1C10, and DE7, and had affinities for AFG1 of 5.1 x 10(7), 2.7 x 10(8), and 1.1 x 10(8) liters mol(-1), respectively.
... Vassilieff, 1997b; Ripley et al., 2001) and body fluids (Leng et al., 1996;Shan et al., 1999) gives an indication with respect to the extent of exposure. It is known that fenvalerate is highly lipophilic and may permeate adipose tissue, testis and ovarian follicular fluid (Chen et al., 2005). ...
The aim of this study was to investigate the potential estrogenic activity of fenvalerate by examining reproductive and fertility capabilities in Wistar rats. Adult male animals were treated for 30 d with 20 or 40 mg/kg/d fenvalerate or corn oil (vehicle) by oral gavage. Further, a possible estrogenic activity of fenvalerate (0.4, 1, 4, 8, or 40 mg/kg) was tested after a 3-d treatment of immature female rats using the uterotrophic assay. Exposure to the higher dose of fenvalerate was toxic to testis and epididymis as shown by a decrease in the absolute weights and sperm counts in both organs. Although the sperm counts were reduced, the fertility and sexual behavior were similar in control rats and rats treated with 40 mg/kg pesticide. Fenvalerate did not exert estrogenic activity in vivo at the tested doses. Data suggest that fenvalerate treatment in this study failed to compromise fertility, possibly due to enhanced reproductive capacity in rodents compared to humans.
... Immunization and Antiserum Preparation. Permethrin antisera were obtained following the protocol reported earlier (Shan et al., 1999b). Briefly, for each immunogen (4-Thyr, 5-Thyr), two New Zealand white rabbits were immunized (rabbits 546 and 548 for 4-Thyr and rabbits 549 and 550 for 5-Thyr). ...
Permethrin is a predominant pyrethroid widely used in agriculture and public health. A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of permethrin was developed. Two haptens, the trans- and cis-isomers of 3-(4-aminophenoxy)benzyl-3-(2, 2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The resulting ELISA has an I(50) value of 2.50 microg/L and relatively low cross-reactivities with other major pyrethroids, such as esfenvalerate, cypermethrin, deltamethrin, and cyfluthrin. Methanol was found to be the best solvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters are unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths (>0.2 M PBS) strongly suppress the absorbances. River water samples fortified with permethrin were analyzed according to this method and validated by GC-MS. Good recoveries and correlation with spike levels were observed, suggesting this immunoassay is valuable for environmental monitoring and toxicological studies at parts per trillion levels of permethrin.
... These methods are relatively time-consuming and expensive and not particularly suitable for large numbers of samples. An immunoassay would provide a sensitive, selective, and rapid method for the detection of this pyrethroid at trace levels (16)(17)(18)(19)(20)(21)(22). For the purpose of raising antibodies that have selectivity for cypermethrin, the cypermethrin molecule must be modified to attach a spacer arm containing a functional group to facilitate coupling to a carrier protein. ...
A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of cypermethrin was developed. Two haptens, the trans- and cis-isomers of 3-[(+/-)-cyano-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyloxy]methyl]phenoxyacetic acid, were conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for cypermethrin was optimized and characterized. The IC(50) for cypermethrin was 13.5 +/- 4.3 microg/L, and the lower detection limit (LDL) was 1.3 +/- 0.5 microg/L. This ELISA had relatively low cross-reactivities with other major pyrethroids, such as deltamethrin, phenothrin, resmethrin, fluvalinate, and permethrin. Methanol was found to be the best organic cosolvent for this ELISA, with an optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was applied to various domestic and environmental water samples. The water samples, fortified with cypermethrin, were analyzed according to this method. Good recoveries and correlation with spike levels were observed.
... Because the target DCCA-glycine is of a small molecular weight (MW 265) and requires conjugation to carrier proteins in order to be immunogenic, various haptens with the carboxylic group or amine group were synthesized. The reactions followed the procedures used in previous publications (32,37). ...
Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine (hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-l-phenylalanine (hapten 5), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis) between antibody and coating antigen showed a high sensitivity for the target analyte. The IC50 was 1.3, 2.1, and 2.2 microg/L for the trans-target analyte and 0.4, 2.3, and 2.8 microg/L for the cis-target analyte. The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite (DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total cis/trans-DCCA-glycine in human urine samples.
... A modified Beilstein method can detect pyrethroid residues on bednets (Muller, 1994;Verle, 1998), but the method is not quantitative and lacks specificity in detecting pyrethroid insecticides on coloured bednets (Drakeley et al., 1999). Chromatographic (especially HPLC) and immunological methods are available for the detection of insecticide residues Beach et al., 1993;Lindsay et al., 1991b;Shan et al., 1999aShan et al., , 1999bWengatz et al., 1998) but they are technically demanding and do not meet the requirements for an easy, inexpensive field test that could be performed by inexperienced staff in the framework of ITN programmes. ...
... Food Chem., Vol. 54, No.23, 2006 Zeng et al. ...
Accurate quantification of quizalofop-p-ethyl is essential for it may do harm to humans and animals through both water and food. Currently, detection of quizalofop-p-ethyl mainly relies on methods such as gas chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry. Although these techniques are reliable, they are relatively expensive and time-consuming because of multistep sample cleanup. To address this, we developed a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with a polyclonal antibody against quizalofop-p-ethyl that was generated in our lab. The IC(50) of detection was 0.03495 microg/mL, and the lowest detection limit reached 0.00192 microg/mL. Furthermore, the method had high specificity for it did not cross-react with other structure-related compounds. When water and soil samples that were fortified with quizalofop-p-ethyl were analyzed by this ELISA, recoveries were in the range of 89-110% from water and 81-108% from soil. Good correlations between this immunoassay and gas chromatography data were obtained for residues of quizalofop-p-ethyl in water and soil. Our data indicate that this method is a convenient analytical technique for monitoring quizalofop-p-ethyl in waters without extraction and the extra cleanup step and in soil without the cleanup step.
... Immunoassay has proven to be a sensitive analytical method for clinical diagnostics, agriculture, environmental monitoring, and food quality assessment. Previously we reported several immunoassays for the detection of pyrethroid parent compounds (23)(24)(25)(26)(27), their primary metabolites, and various conjugates (28)(29)(30). Although no study has specifically determined the exact † University of CaliforniasDavis. ...
Pyrethroids are widely used in agriculture as insecticides. This study describes a sensitive enzyme-linked immunosorbent assay for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Four antisera were elicited against two different immunizing haptens. Antisera were characterized in combination with several coating haptens. The lowest IC50 value (0.5 ng/mL) was obtained with antiserum 1891 and 3-phenoxybenzoic acid-BSA conjugate as the coating antigen. Antiserum 1891 was highly selective for the target compound with an overall cross-reactivity of <0.3% to structurally related compounds. The assay sensitivity was negligibly affected by pH 4-9. A 5-fold improvement in IC50 was observed using a 10-fold concentrated phosphate-fuffered saline as the assay buffer. Compared to assays conducted in normal phosphate-fuffered saline, the maximal absorbance was almost identical. A good correlation (r 2 = 0.99 and 0.97 for urine samples A and B, respectively) was observed between spiked levels and the levels detected by the immunoassay.
This study aimed to investigate the effect of pyrethroid insecticides on the regulation of bovine cervical function. Cervical cells or strips obtained from cows during the periovulation period were treated with cypermethrin and fenvalerate (0.1-10 ng/ml). None of the pyrethroids exerted a cytotoxic effect, whereas only fenvalerate increased the cervical contraction force and mRNA expression of receptor of oxytocin (ROT) and prostaglandin (PG) synthases. Both pyrethroids inhibited PG secretion and decreased the amount of diacylglycerol (DAG), which is the second messenger involved in oxytocin signal transmission, and fenvalerate decreased the myosin light-chain kinase (MLCK) level. These finding indicate that fenvalerate induces greater disruption of cervical function than cypermethrin.
1. Non-essential heavy metals such as mercury (Hg), arsenic (As), cadmium (Cd), and aluminium (Al) are useless to organisms and have shown extensive toxic effects. Previous studies show that two main molecular mechanisms of metal toxicity are oxidative stress and metal-metal interaction which can disrupt metal homeostasis.
2. In this paper, we mainly illustrate metal toxicity and metal-metal interaction through examples in mammalians and D. melanogaster (fruit fly).
3. We describe the interference of metal homeostasis by metal-metal interactions in three aspects including replacement, cellular transporter competition, and disruption of the regulation mechanism, and elaborate the mechanisms of metal toxicity to better deal with the challenges of heavy metal pollution and related health problems.
Insecticides are natural and synthetic chemicals used to kill unwanted pests. However, humans and insect share similar molecular targets, and thus, insecticides are potentially hazardous to human health. Several health effects might be observed in experimental animals following controlled exposure to insecticides. Synthetic pyrethroids are still a relatively novel group of insecticides widely used not only in agriculture but also in human and veterinary medicine, forestry, and public health and for commercial pest control and residential consumer use. They play a unique role in fighting against malaria in tropical areas, where the WHO recommends pyrethroids among others for indoor residual spraying (IRS) and impregnation of bed nets to prevent mosquito biting.
The title compound (R)-N′-[2-(4-methoxy-6-chloro)-pyrimidyl]-N-[3- methyl-2-(4-chlorophenyl)butyryl]-urea has been synthesized, and its crystal structure and biological behaviors were studied. Crystallographic data: C 17H18Cl2N4O3, M r = 397.25, monoclinic, space group P21/c, a = 12.331(2), b = 14.025(3), c = 23.085(5) Å, β = 99.607(4)°, Z = 8, V = 3936.2(13) Å3, Dc = 1.341 g/cm3, F(000) = 1648, R = 0.0718, wR = 0.1585 and μ(MoKα) = 0.353 mm-1. The preliminary biological tests showed that the title compound has definite insecticidal and fungicidal activities.
A sensitive, automated, non-competitive fluorescent immunoassay was developed for quantitative analysis of 3-phenoxybenzoic acid (PBA) in human urine sampes as a putative biomarker of exposure to pyrethroid insecticides using the bench top immunoanalyzer, KinExA™ 3000 system. The key difference between the KinExA system and the enzymelinked immunosorbent assay (ELISA) is to eliminate the PBA-antibody interaction with the coating antigen. This can be achieved by separately capturing the free PBA-antibody onto the hapten-immobilized beads when a constant amount of reaction solution in equilibrium between PBA-antibody and analyte passes through the bead-packed glass capillary column. Optimal dilution of the PBA antibody was determined when fluorescent signals of 0.5-2 were obtained and a sufficient amount of coating antigen was immobilized to ensure the capture of all free antibodies. IC 50S of the two KinExA methods (0.3 and 0.6 ng/mL for one- and two-step KinExA, respectively) were 3- and 6-fold better than the heterologous ELISA and were approximately 650- and 300-fold lower compared to that of the homologous ELISA (IC 50 of 200 ng/mL). The KinExA assay was negligibly affected within tested range of pHs (5-10) and ionic strengths (1, 5, and 10X PBS). Similar urine matrix effects were observed in the two KinExA assays with a 5- to 10-fold increase in IC 50s when 5 and 10% of urine was contained in the reaction buffer. A high correlation (r 2 = 0.99) was observed between detected and spiked concentrations of PBA standard with average recoveries of 88-160%.
Monitoring studies for toxic substances in environmental and biological samples are essential for evaluating risk to human and environmental health. Immunoassays are one method that can satisfy analytical demands for high sample loads requiring high sensitivity because they are rapid, can be adapted to high throughput or biosensor formats and can be adapted for small sample sizes. A description of progress in the field of immunoassays and the application to biosensors in general is presented with a focus on research from this laboratory.
This study aimed at developing a method for the preparation of hapten and immunogen for producing polyclonal antibody specific to cypermethrin for the determination of insecticide residues in contaminated fruits and vegetables. 3-(2, 2-dichlorovinyl)-2, 2-dimethyl - (1-cyclopane) carboxylic acid (DCCA) was used as a spacer arm of hapten preparation. DCCA was linked to beta alanine methyl ester and conjugated with bovine serum albumin (BSA) as an immunogen and with oval albumin (OVA) as a coating antigen. BALB/c mice were immunized with immunogen. After three doses, sera from each mouse were collected and used to develop an ELISA. 0.05% Tween20/PBS with 20% methanol was used as diluents. The obtained polyclonal antibody (pAb) gave IC50 to cypermethrin at 5.2 μg/well. The lowest quantification limit of detection of the ELISA was 0.18 μg/well at 85% B/B0 and the detection limit was 0.039 μg/well at 90% B/B0. The antibody will be applicable for developing ELISA for screening of cypermethrin residue in vegetable and fruit samples and enable to save cost of currently used analytical procedures.
An immuno multiresidue screening assay in microarray format for the determination of complex chemical mixtures at the microgram per liter level, using antibody-functionalized gold nanoparticles, is presented. The analytical method relies on the use of a cocktail of nanogold-labeled specific antibodies, acting as recognition and detection species. The concept of multireside screening is proved by developing a multiplex assay on a compact disk support for the determination of 2-(2,4,5-trichlorophenoxy)propionic acid, 3-phenoxybenozic acid, 4-nitrophenol, alachlor, atrazine, azoxystrobin, chlorpyrifos, diazinon, diuron, endosulfan, fenthion, forchlorfenuron, imidacloprid, malathion, pentachlorophenol, pyraclostrobin, sulfasalazine, and triclosan, achieving detection limits of 0.07, 0.24, 10.9, 0.21, 0.14, 0.11, 0.11, 102, 0.36, 1.8, 1.7, 0.06, 0.08, 5.8, 1.0, 0.39, 0.003, and 12 μg/L, respectively. Due to the selectivity of the antibody-functionalized nanoparticles, the developed screening methodology allows the simultaneous determination of mixtures of water pollutants in a 10-plex configuration. The analytical performances were compared with those of reference chromatographic methods by the analysis of spiked water samples, the sensitivity and recovery results being in good agreement. The presented screening approach directly quantifies the concentration of complex chemical mixtures without sample treatment or preconcentration steps in a total time of 35 min.
An enzyme-linked immunosorbent assay (ELISA) was developed to detect organophosphorus pesticides using a phage-borne peptide that was isolated from a cyclic 8-residue peptide phage library. The IC50 values of the phage ELISA ranged from 1.4 to 92.1 µg L–1 for eight organophosphorus pesticides (parathion-methyl, parathion, fenitrothion, cyanophos, EPN, paraoxon-methyl, paraoxon, fenitrooxon). The sensitivity was improved 120- and 2-fold compared to conventional homologous and heterologous ELISA, respectively. The selectivity of the phage ELISA was evaluated by measuring its cross-reactivity with 23 organophosphorus pesticides, among which eight were the main cross-reactants. The spike recoveries were between 66.1% and 101.6% for the detection of single pesticide residues of parathion-methyl, parathion and fenitrothion in Chinese cabbage, apple and greengrocery, and all of the coefficient of variation were less than or equal to 15.9%. Moreover, the phage ELISA results were validated by gas chromatography. The results indicate that isolating phage-borne peptides from phage display libraries is an alternative method for the development of a heterologous immunoassay and that the developed assay has a lower limit of detection than the chemically synthesized competitor assay.
Monoclonal antibodies (MAbs) against pyrethroid insecticide fenvalerate were achieved, identified, and applied in environmental water. Mice were immunized with a novel synthesized hapten conjugated with bovine serum albumin (BSA). Three positive clones of MAbs were obtained after cell fusion and hybridoma selection, among them MAb-2 (5B10) showed the highest reactivity toward fenvalerate. The IC50 of MAb-2 was 94.5 ng mL-1; moreover, it showed lower cross-reactivity with other pyrethroids such as bifenthrin, tetramethrin, deltamethrin and beta-cypermethrin. Optimization of enzyme-linked immunosorbent assay (ELISA) was studied. The limit of detection (LOD) of the assay was 8.8 ng mL-1 and the detection range was 0.017-27.33 g mL-1. For preliminary application, addition recovery experiments in water samples were performed. The mean recoveries of three kinds of samples varied from 90.6% to 108.7% and the coefficients of variation ranged from 0.5% to 5.3%. The results showed that MAb-2 could be used for the detection of fenvalerate contamination in environmental water.
Two kinds of new immunogens and coating antigens had been designed and synthesised by methods of lively ester (DCC) and carbodiimide (EDC), respectively. Preliminary immunosorbent assays indicated that the two target conjugates presented good inhibition ratio against the haptens of cypermethrin in vivo. The results showed that the best efficient titres of anti-cypermethrin rabbit serum in methods of DCC and EDC were 1:32000 and 1:64000, the IC50 of them were 18.907µg/L and 16.877µg/L against the haptens of cypermethrin, respectively. The data indicated that the anti-cypermethrin antibodies in method of EDC had higher sensitivity and higher selectivity than the anti-cypermethrin antibodies in method of DCC. Thus, we can synthesise antigens and anti-bodies with method of EDC. The antibodies of high quality can be effectively used in the field of the rapid detection of pyrethroid pesticide residues in the research of the reagent strips.
Immunoassays offer selective, sensitive and low-cost tools for the assessment of pesticide exposure through the measurement of parent compounds or key metabolites in biological fluids such as urine, blood or saliva. Recently conducted biomonitoring studies for paraquat and atrazine illustrate the strengths and weaknesses of the immunochemical approach. Development for improved assay throughput and sensitivity includes substitution of enzyme labels with fluorescent nanoparticle probes or luminescent acridinium labels together with the use of automated immunoanalyzers, immunosensors or microchips with flow-through systems.
Pyrethroids are widely used in agriculture as insecticides. In this study, we describe a simple one-step homogeneous fluoroimmunoassay for the glycine conjugate of phenoxybenzoic acid (PBAG), a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Quenching fluoroimmunoassay (QFIA) is based on the competition of labeled and non-labeled pesticide for binding with antibodies and the resulting calibration curve is based on the relationship between analyte concentration and fluorescence quenching of labeled pesticide by specific antibodies. We developed a QFIA for PBAG in aqueous solution using fluorescein-labeled PBAG and polyclonal antibodies. The estimated IC50 (analyte concentration giving 50% inhibition of quenching) for PBAG was 4.5nM. The detection limit (DL) was 0.9nM. The dynamic range of the calibration curve was 2–50nM. The average analytical recovery obtained by applying the method to urine samples (400- or 1000-fold urine dilution) was 85–111%. This demonstrates the QFIA to be a very simple and rapid detection method for PBAG; no washing steps and no enzyme conjugates were required.
An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for cyhalofop-butyl was developed with a polyclonal antibody produced against a hapten (cyhalofop acid) conjugated with bovine serum albumin (BSA). The ELISA of cyhalofop-butyl showed an IC50 value of 0.067±0.004mg/l and the limit of detection (LOD, IC10) of 0.0029±0.0001mg/l at the optimal conditions. No significant cross-reaction to other structure-related compounds suggested high specificity for cyhalofop-butyl of the method. The average recoveries of cyhalofop-butyl from fortified water and soil were in the range of 83.2–119.7% and 80.1–104.0%, respectively. These data indicate that this method is a convenient analytical technique for monitoring cyhalofop-butyl in water and soil without purification steps.
The aim of this study was to identify a plasma biomarker of exposure to pyrethroid insecticides. A major metabolite, 3-phenoxybenzoic acid (3-PBA), can be detected in urine but urinary 3-PBA cannot be used to assess the active dose. The 3-PBA-adduct represents a much more persistent class of biomarkers than metabolites excreted into urine, having half lives up to several weeks or months. We developed an enzyme-linked immunosorbent assay (ELISA) for total 3-PBA including adduct formed after alkaline hydrolysis, liquid-liquid extraction (LLE) and solid phase extraction (SPE) of the sample. The developed ELISA had an IC50 value of 26.7 ng/mL. The intra- and inter-assay coefficients of variation (%CV) were lower than 5% and were within the optimum condition variance (OCV) range. The LLE cleanup technique satisfactorily eliminated the matrix effect from plasma samples before SPE and ELISA analysis yielding good recoveries (85.9-99.4%) with a limit of quantitation (LOQ, 5 ng/mL) that was 30- to 47-fold more sensitive than previous studies. Moreover, the developed method could separate more than 80% of 3-PBA from adduct form. The method was successfully applied to the detection of the target in real samples obtained from consumers (n=50) and farmers (n=50). To our knowledge, this is the first ELISA method for detecting 3-PBA in human plasma and applied to a field study.
Two series of haptens including 3-phenoxybenzoic acid (PBA) and 3-(2-chloro-3, 3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclo-propanecarboxylic acid (CF3MPA) were used to prepare immunogens through attachment of 4-C or 6-C handles. Class selective antibodies were produced by immunising rabbits. Ab502 showed the highest reactivity towards tau-fluvalinate (IC50 1.3 ng mL-1), λ-cyhalothrin (IC50 2.3 ng mL-1), cyfluthrin (IC50 2.2 ng mL-1) and fenpropathrin (IC50 18.5 ng mL-1) among the antibodies in a competitive ELISA. The effects of methanol, pH and salt concentration were optimised for maximum efficiency of the ELISA (Enzyme-Linked ImmunoSorbent Assay). Ab502 (1:80000)/2-OVA-1(0.2 µg mL-1) was chosen for ELISA optimisation. Finally, 0.05 M phosphate buffered saline (PBS) at pH 6.5 containing 30% methanol (v/v) was used to dilute the standards. Target analytes in honey samples were extracted with ethyl acetate by sonication. The samples were spiked with three different concentrations of each compound (tau-fluvalinate, 0.5 ng g-1, 3 ng g-1, 12 ng g-1; λ-cyhalothrin and cyfluthrin 1 ng g-1, 5 ng g-1, 65 ng g-1). The recoveries were 36-59% at the lowest spiking concentration and 61-81% at the higher concentration. This assay might be useful to screen pyrethroid residues in honey or other matrix.
A novel method was developed for the determination of eight pyrethroids in water samples by liquid–liquid microextraction
based on solidification of floating organic droplets followed by gas chromatography with electron capture detection. The type
and volume of the extraction solvents, extraction time, sample solution temperature, stirring rate and ionic strength were
studied and optimized. Under the optimum conditions, enrichment factors ranged from 824 to 1,432, and the limit of detection
range from 2.0 to 50ng∙L−1. The calibration graph is linear from 0.15 to 80μg∙L−1 for cyfluthrin, fenvalerate, fluvalinate and deltamethrin, 0.09 to 80μg∙L−1 for fenpropathrin, 0.006 to 80μg∙L−1 for lambda-cyhalothrin, 0.026 to 80μg∙L−1 for permethrin, 0.01 to 80μg∙L−1 for cypermethrin. The correlation coefficients (r) varied from 0.9961 to 0.9988. The method was successfully applied to the determination of pyrethroid pesticide residues
in tap water, well water, reservoir water, and river water. Recoveries ranged from 79.0% to 113.6%, and relative standard
deviations are between 4.1% and 8.7%.
KeywordsPyrethroids-GC-ECD-Water samples-Liquid–liquid microextraction based on solidification of floating organic droplet
The peptide P7, with an amino acid sequence of Cys-Val-Thr-Pro-Ala-Leu-Arg, inhibits angiotensin I-converting enzyme (ACE).
In this paper, P7 was identified in peanut protein isolate (PPI) and peanut polypeptides (PPs) with a new method. The P7 peptide
was synthesized and used in the preparation of an antiserum. Using the antiserum, P7 was specifically identified in PPI by
western blot, and its level in PPI and PPs was assayed by dot blot hybridization. The results showed that two bands of P7
expression with molecular weight 18–25 and 25–35kDa were seen in PPI by glucan gel chromatography. The positive reaction
rate of P7 in PPs was higher than in PPI, consistent with the measured ACE inhibitory activity. The rate of P7 in sample no.
3 reached 39.29% of the positive control, using a dose of 20μg/mL. This sample had an ACE inhibitory activity of 89.73%.
Therefore, western blot and dot blot hybridization with prepared antibody against synthetic peptide was a very sensitive detection
method for peptide.
Two solid-phase extraction (SPE) methods, based on hydrophobic and selective (antibody–antigen) interactions, have been established and evaluated as clean-up methods prior the immunochemical analysis of 2,4,6-trichlorophenol (2,4,6-TCP) in urine samples. Without a clean-up method the extent of interferences caused by the urine matrix in the ELISA [R. Galve, M. Nichkova, F. Camps, F. Sanchez-Baeza, M.-P. Marco, Anal. Chem. 74 (2002) 468] varies depending on individual urine samples and accurate measurements are only possible when 2,4,6-TCP concentration levels are higher than 40 μg L−1. Both sample preparation methods improve detectability of the immunochemical method getting rid of the variability due to the intrinsic individual differences within the urine samples. Even though, the immunosorbent (IS)-SPE method developed has proven to be a superior sample preparation method eliminating completely matrix effects caused by both, non-hydrolyzed (NH) and hydrolyzed urine samples. The LOD reached by the C18-SPE-ELISA method (∼4 μg L−1 for free and total chlorophenols) is sufficient for exposure assessment of the occupationally exposed population. However, the detectability (0.66 and 0.83 μg L−1 in NH and hydrolyzed urine samples, respectively) accomplished by the IS-SPE-ELISA allows also biomonitoring potential exposure of non-occupationally exposed groups. Moreover, the specificity of the IS-SPE procedure can be modulated to provide a group-specific (9 chlorophenols and 2 bromophenols are extracted with an efficacy superior to 85%) or a more selective protocol (only 2,3,4,6-TtCP, 2,4,6-TCP are extracted with a recovery superior to 80% and 2,4,6-tribromophenol with a 70% recovery). On the other hand, the IS-SPE extracts produce cleaner chromatograms allowing quantitation by GC–ECD (or GC–MS) after toluene extraction and derivatization with a LOD near 0.1 μg L−1 in NH and hydrolyzed urine samples. The IS-SPE-ELISA method has been validated with GC–ECD using spiked and real urine samples. This study also provides evidences of the general exposure of the population to organochlorinated and organobrominated substances. Measurable levels of 2,4,6-TCP, 2,4,5-TCP, 2,3,4,6-TtCP, 2,4,6-TBP and 2,4-DBP have been detected in some of the samples used in this study.
A sensitive and broad class selective direct competitive enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (McAb) has been described for the detection of pyrethroids with phenoxybenzene group. One monoclonal antibody, 2G(2)E(7), was obtained and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice. The assay with the most selectivity for the family pyrethroids with phenoxybenzene group was optimized. The IC(50) values of the optimized immunoassay were 1.8 μg L(-1) for deltamethrin, 1.5 μg L(-1) for cypermethrin, 2.0 μg L(-1) for fluvalinate and fenvalerate, 2.2 μg L(-1) for phenothrin, 2.4 μg L(-1) for flucythrinate, 3.0 μg L(-1) for fenpropathrin, and 5.0 μg L(-1) for permethrin. River water samples fortified with pyrethroids were analyzed with the ELISA to evaluate the accuracy of the assay. The recoveries of pyrethroids in spiked water samples ranged from 74 to 108%. The results indicate that the ELISA developed can accurately simultaneously determine pyrethroids with phenoxybenzene group in water samples.
The antibacterial soap additive triclocarban (TCC) is widely used in personal care products. TCC has a high environmental persistence. We developed and validated a sensitive online solid-phase extraction-LC-MS/MS method to rapidly analyze TCC and its major metabolites in urine and other biological samples to assess human exposure. We measured human urine concentrations 0-72 h after showering with a commercial bar soap containing 0.6% TCC. The major route of renal elimination was excretion as N-glucuronides. The absorption was estimated at 0.6% of the 70±15 mg of TCC in the soap used. The TCC-N-glucuronide urine concentration varied widely among the subjects, and continuous daily use of the soap led to steady state levels of excretion. In order to assess potential biological effects arising from this exposure, we screened TCC for the inhibition of human enzymes in vitro. We demonstrate that TCC is a potent inhibitor of the enzyme soluble epoxide hydrolase (sEH), whereas TCC's major metabolites lack strong inhibitory activity. Topical administration of TCC at similar levels to rats in a preliminary in vivo study, however, failed to alter plasma biomarkers of sEH activity. Overall the analytical strategy described here revealed that use of TCC soap causes exposure levels that warrant further evaluation.
This paper describes some of the early work on pyrethroid insecticides in the Casida laboratory and briefly reviews the development and application of immunochemical approaches for the detection of pyrethroid insecticides and their metabolites for monitoring environmental and human exposure. Multiple technologies can be combined to enhance the sensitivity and speed of immunochemical analysis. The pyrethroid assays are used to illustrate the use of some of these immunoreagents such as antibodies, competitive mimics, and novel binding agents such as phage-displayed peptides. The paper also illustrates reporters such as fluorescent dyes, chemiluminescent compounds, and luminescent lanthanide nanoparticles, as well as the application of magnetic separation, and automatic instrumental systems, biosensors, and novel immunological technologies. These new technologies alone and in combination result in an improved ability to both determine if effective levels of pyrethroids are being used in the field and evaluate possible contamination.
A general and broad class selective competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the alpha-cyano pyrethroids was developed. One class specific hapten, the 5-(3-benzylphenyl)-5-cyanopentanoic acid (CPBA-BE), was conjugated with BSA as an immunogen. Polyclonal antibodies, PY-antibodies, were generated. The assay with the most selectivity for the family alpha-cyano pyrethroids such as cyphenothrin was further optimized and tested for tolerance to cosolvent, pH, and ionic strength changes. The IC(50) values of the optimized immunoassay were 4.58 microg L(-1) for cyphenothrin, 5.62 microg L(-1) for fenpropathrin, 7.08 microg L(-1) for deltamethrin, 10.72 microg L(-1) for cypermethrin, 19.95 microg L(-1) for flucythrinate, and 28.18 microg L(-1) for esfenvalerate. The cross-reactivities of cyphenothrin, fenpropathrin, deltamethrin, cypermethrin, flucythrinate, and esfenvalerate were 100%, 81.49%, 64.68%, 42.72%, 22.96%, and 16.25%, respectively. The low detection limit (LDL) of this assay was 0.107 microg L(-1) for cyphenothrin. This method was used for the detection of the alpha-cyano pyrethroids in Tai lake water.
Chlorophenols are frequently found in the urine of the population as consequence of the widespread use of chlorophenols and other organochlorinated compounds. An immunoassay for 2,4,5-trichlorophenol (2,4,5-TCP) has been evaluated as a tool to assess risk exposure of the population to these substances. The immunoassay is stable in media with pH values ranging from 6.6 to 10.5 units and ionic strength values varying within 20 and 80 mS cm(-)(1). Considering these parameters, the optimized immunoassay shows a limit of detection of 0.05 microg L(-)(1) and the dynamic range is placed between 0.09 and 0.72 microg L(-)(1). It shows a good accuracy and the coefficients of variation within and between assays are around 12% or lower. However, matrix effects can diminish the efficiency and detectability of the immunochemical methods. In this paper, the effect of water and complex biological sample matrices, such as serum and urine, on the immunoassay for 2,4,5-TCP has been evaluated. Simple sample treatment procedures have been developed for the analysis of these matrices. The final analytical protocols allow straightforward immunochemical determination of 2,4,5-TCP in natural waters, urine, and serum with detection limits of 0.07, 0.26, and 0.8 microg L(-)(1), respectively.
The pyrethroids are one of the most heavily used insecticide classes in the world. It is important to develop sensitive and rapid analytical techniques for environmental monitoring and assessment of human exposure to these compounds. Because major pyrethroids contain a phenoxybenzyl group and phenoxybenzoic acid (PBA) is a common metabolite form or intermediate, PBA might be used as a biomarker of human exposure to pyrethroids. A sensitive and selective immunoassay for the common pyrethroid metabolite PBA was developed. Rabbits were immunized with 3-[4-(3-carboxyphenoxy)phenoxy] N-thyroglobulin ethylamine. All sera were screened against numerous coating antigens. The assay with the least interference and the best sensitivity was optimized and characterized. The average IC50 for free PBA was 1.65 ng/mL. No cross-reactivity was measured to parent pyrethroids and other metabolites. Urine matrix effects can be eliminated by simple dilution. Results from urine samples from exposed workers suggest that this PBA immunoassay might be suitable as a monitoring tool for human exposure to pyrethroids.
A recombinant glutathione S-transferase (GST)-based pyrethroid quantification assay was field-tested in Ifakara, Tanzania. Initial laboratory tests suggested that all reagents used in the assay should be sufficiently stable for field use, provided that domestic refrigeration facilities were available. Insecticide-impregnated bednets were collected from a region where a social marketing programme was in progress. A total of 100 bednets were collected and the assay plus standard HPLC analysis was performed on the residues extracted from four replicate areas of each net. Insecticide residue estimations for assays performed on white and pale green bednet samples were accurate when compared with residue analysis by HPLC. However, for dark green or blue bednets, there was no correlation between the GST-based assay and HPLC pyrethroid quantification results. The assay failure with the dark coloured nets was caused by the extraction of the dyes along with the insecticide, which subsequently interfered with the GST assay. When the same samples were analysed by HPLC, the dyes were separated from the insecticide by reverse phase column chromatography and hence did not affect the results.
A new rapid and sensitive electron ionization gas chromatography-mass spectrometry method in selective ion monitoring mode (SIM) was developed for the determination of l3 synthetic pyrethroid insecticide molecules and their stereo isomers in whole blood. The pyrethroid insecticides investigated are allethrin, bifenthrin, cypermethrin, cyphonothrin, cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, fenpropathrin, imiprothrin, permethrin, prallethrin and transfluthrin. The residues of pyrethroids are extracted from the whole blood using hexane and acetone mixture (80 + 20%) as solvent. All the pyrethroid residues were separated by using a gas chromatography-mass spectrometry operated in electron ionization mode and quantified in selective ion monitoring mode. The method can detect the residues of different pyrethroids down to the level 0.05-2 ng/ml. Recovery experiments conducted in whole blood samples at the fortification level 1-1000 ng/ml showed 91-103% recovery. The applications of the analytical method for the determination of pyrethroid residues in real samples were tested by analyzing 45 human blood samples collected from the population exposed continuously to different pyrethroid based formulations. The results are confirmed by spiking the known quantity of pyrethroids and subsequently their positive detection.
Estrogens, whether natural or synthetic, clearly influence reproductive development, senescence, and carcinogenesis. Pyrethroid insecticides are now the most widely used agents for indoor pest control, providing potential for human exposure. Using the MCF-7 human breast carcinoma cell line, we studied the estrogenic potential of several synthetic pyrethroid compounds in vitro using pS2 mRNA levels as the end point. We tested sumithrin, fenvalerate, d-trans allethrin, and permethrin. Nanomolar concentrations of either sumithrin or fenvalerate were sufficient to increase pS2 expression slightly above basal levels. At micromolar concentrations, these two pyrethroid compounds induced pS2 expression to levels comparable to those elicited by 10 nM 17ss-estradiol (fivefold). The estrogenic activity of sumithrin was abolished with co-treatment with an antiestrogen (ICI 164,384), whereas estrogenic activity of fenvalerate was not significantly diminished with antiestrogen co-treatment. In addition, both sumithrin and fenvalerate were able to induce cell proliferation of MCF-7 cells in a dose-response fashion. Neither permethrin nor d-trans allethrin affected pS2 expression. Permethrin had a noticeable effect on cell proliferation at 100 microM, whereas d-trans allethrin slightly induced MCF-7 cell proliferation at 10 microM, but was toxic at higher concentrations. Overall, our studies imply that each pyrethroid compound is unique in its ability to influence several cellular pathways. These findings suggest that pyrethroids should be considered to be hormone disruptors, and their potential to affect endocrine function in humans and wildlife should be investigated.
The impetus for this book came from numerous requests by public and private agencies and citizens for information regarding the human health effects of pes ticide exposures. We have tried to compile a relatively complete, concise sum mary of the acute and chronic health effects and the toxicology of pesticides in a format that provides quick and easy access. This book was written to address the needs of the following groups: medical and public health professionals, tox icologists, environmentalists, industrial hygienists, regulators, producers and users of pesticides, public interest advocates, and the legal profession. Acknowledgments We are indebted to Mr. Christopher J. Wiant, Chief of the Environmental Chemistry Section of the Illinois Department of Public Health. The financial support provided by his office was essential in producing this book. We are also indebted to Dr. Charles Benbrook, former staff member, and Representative George E. Brown, Chairman of the Subcommittee on Depart ment Operations, Research and Foreign Agriculture of the Committee on Agri culture, United States House of Representatives, for their guidance in obtain ing pesticide toxicity data. In the Freedom of Information Office, Office of Pesticide Programs of the United States Environmental Protection Agency, the patience and assistance of Therese Murtagh and Virginia Salzman in obtaining documents are appreciated. Of the numerous individuals who participated in the production of this book, the following merit special recognition for the quality of their research, editing, and critical skills: Mark Loafman, Sue Ramirez, Steve Smith, Sally Burns, and Denise Steurer.
The relative importance of detoxification and target site specificity in determining pyrethoid structure-activity relationships is considered with three examples, i.e.: houseflies and rethronyl chrysanthemates and analogs with various unsaturated side chains in the acid and alcohol moieties affecting the ease of synergism by piperonyl butoxide; American cockroaches and the trans- and cis isomers of tetramethrin, phenothrin and permethrin of high potency for inducing repetitive discharges in the cercal sensory nerve but not always of parallel toxicity; mice and the trans- and cis isomers of resmethrin, ethanomethrin, phenothrin and cyphenothrin varying from exceptionally low to moderately high intraperitoneal and intracerebral toxicity. There are large differences in the contribution of esterases and oxidases to detoxification and in the optimal synergist depending on the pyrethoid and species involved. Recent studies on pyrethroid metabolic chemistry have identified 12 conjugates of 3-phenoxybenzoic acid in plants, insects, birds and 13 species of mammals and three bile acid conjugates of the fluvalinate acid moiety in chickens, rats and cows. trans-Tetramethrin is decarboxylated via the epoxychrysanthemate and undergoes reduction of the tetrahydrophthalimide fragment possibly via the glutathione conjugate. Several of the fluvalinate and flucythrinate metabolites are now identified. Derivatizations of the oxime ether and dihalovinyl substituents of potent pyrethroids provide the first examples of propyrethroids undergoing bioactivation.
Few discussions in the field of immunoassay analysis have centered on the interpretation of immunoassay data. Personnel at Ciba Plant Protection have analyzed several thousand real-world samples by immunoassay and have reached some conclusions from this experience. This paper will discuss the various types of standard curves in use, the means by which the limits of quantitation are determined and the idea of analyte-equivalency to more clearly elucidate the problem of how to interpret immunochemical data. The goal of this paper is to answer the question, "What do the numbers generated by an immunoassay mean?"
On either a single oral dose or 5 consecutive daily doses, the metabolism of fenvalerate [α-cyano-3-phenoxybenzyl-2-(4-chlorophenyl)isovalerate] and the (S)- acid ester isomer in male rats was rapid, and the acid moiety and the aromatic portion of the alcohol moiety were almost completely eliminated from the body within several days. The CN group of the alcohol moiety was rapidly converted mainly to thiocyanate which retained relatively longer in selective tissues including skin and hair. Fenvalerate and the (S)-acid isomer yielded two fecal ester metabolites which resulted from hydroxylation at the 4’- and 2’-phenoxy positions. Other significant metabolites were 3-phenoxybenzoic acid and its hydroxy derivatives (free and conjugates) from the alcohol-labeled compound, 3-(4-chlorophenyl)isovaleric acid and its hydroxy derivatives (free, lactones and conjugates) from the acid-labeled compound, and thiocyanate and CO2 from the CN-labeled compounds. There were no apparent differences in the nature and amount of metabolites, and in the patterns of ¹⁴C excretion and tissue residues between fenvalerate and the (S)- acid isomer. © 1979, Pesticide Science Society of Japan. All rights reserved.
14C標識 esfenvalerate [(S)-α-cyano-3-phenoxybenzyl (S)-2-(4-chlorophenyl) isovalerate] を雌雄ラットおよびマウスに2.5mg/kgまたは2.5mg/kg/dayで1回経口または10日間連続経口投与を行なった. 投与14Cは速やかにかつほぼ完全に尿糞中に排泄された. 14C組織残留量は脂肪以外で全般的に非常に低かった. 主要代謝反応は1) 酸側の2-, 3-位とアルコール側の2′-, 4′-位の酸化, 2) エステル結合の開裂および3) グルクロン酸, 硫酸, グリシンまたはタウリンとの抱合反応であった. 14C-esfenvalerate と fenvalerate [(RS)-α-cyano-3-phe-noxybenzyl (RS)-2-(4-chlorophenyl) isovalerate] の3非標識体 [(2S, αS), (2S, αR) および (2R, αR)] との等量混合物を1回または連続経口投与した. 14C-esfenvalerate の単独投与と比べて14C排泄率, 14C組織残留量および代謝物量に差異は認められなかった. このことは esfenvalerate の体内挙動は他異性体から独立していることを示唆している. また酸側14C標識 fenvalerate とesfenvalerate の代謝を比較すると fenvalerate が全般的に若干高い14C組織残留量を示した. これは fenvalerate だけから生成する cholesteryl (R)-2-(4-chlorophenyl) isovalerate によるもので, この生成以外には esfenvalerate と fenvalerate の生体内運命に大きな差異はなかった.
The chemical properties and metabolism of the synthetic pyrethroids, phenothrin, permethrin, cypermethrin, decamethrin and fenvalenate are reviewed. With the exception of decamethrin, which is a single stereoisomer, all the other pyrethroids discussed are used as a mixture of isomers. These chemicals are all relatively non—volatile; they have very low solubility in water, and can all be hydrolysed at the ester linkage. They are all more stable to photolysis than the natural pyrethroids although phenothrin which contains an isobutenyl group is still relatively photolabile. The photostability of the other chemicals is such that they have been successfully developed as agricultural insecticides. When applied to the leaves of plants, the pyrethroids are not translocated to any significant extent. They are degraded on the plant surface, probably mainly by photodegradation to a wide range of products many of which are formed by ester cleavage and ring hydroxylation. These products are subsequently conjugated by the plant mainly as glycosides. When plants are grown in treated soil, again little translocation of chemical to aerial parts of the plant occurs. That trace of chemical which is translocated is not intact pyrethroid but is metabolites of the acidic half of the original molecule. In soil, the pyrethroids are readily degraded. The tine taken for loss of half of the chemical ranges from about 2-12 weeks depending on the structure of the compound and the soil type. Hydrolysis of the ester linkage is a major degradation route. The products are further modified by loss of the cyanhydrin where applicable and by hydroxylation. The intact pyrethroids can also by hydroxylated. Further degradation also takes place and extensive evolution of 14C02 has been observed from radiolabels incorporated in various positions of the molecules. Most of the degradation observed has been shown to be mediated by soil microorganisms. When dosed to rodents, the pyrethroids are rapidly metabolised and the metabolites are excreted in similar amounts in both the urine and faeces. A large number of metabolites have been identified. These are mainly composed of intact pyrethroids and hydrolysis products both of which may be hydroxylated in various positions. The metabolites may also be conjugated with sulphate glucuronide, taurine and other chemicals. In addition radiolabelled permethrin has been bsed to ruminants and hens. Again excretion was rapid and only small amounts of dosed radioactivity transferred to the meat, milk and eggs of the treated animals. Sensitive residue analysis techniques are available for the analysis of the pyrethroids. In general the methods involve an initial extraction with organic solvent, followed by a liquid-liquid partition and an adsorption chromatography ‘clean-up’ step. The final determination is by gas–liquid chromatography using an electron capture detector. Various gic columns may be used to give total or partial separation of the isomers or to avoid separation of isomers. In the case of phemothrin, due to its poor sensitivity to an EC detector, analysis procedures are itore complex. No publications are available describing analysis of the breakdown products of the pyrethroids but some of the methods suggested for parent compounds involve a hydrolysis step and these could be adapted for analysis of metabolites.
The development of an enzyme-linked immunosorbent assay (ELISA) for the herbicide molinate (Ordram, S-ethyl hexahydroazepine-1-carbothioate) is described. By a thiol replacement reaction of thiocarbamate sulfones, several haptens were synthesized. These haptens were coupled to carrier proteins via mixed-anhydride and diazotization procedures. Antibodies raised against these antigens were screened for sensitivity and specificity for molinate. An assay is described with a limit of detectability of 3 ng/mL that is specific for molinate with some cross-reactivity (15%) to molinate sulfone. Other thiocarbamate pesticides had cross-reactivities of <1.4%. Propylene glycol, as a carrier solvent, interfered in the ELISA at concentrations of 12.5% and greater, whereas acetonitrile had no effect at 12.5% but had increased and then decreased absorbances relative to control at 5% and 25%, respectively. The half-lives of molinate in a laboratory study as measured by liquid scintillation counting and ELISA were 2.6 and 2.2 days, respectively. Paired t statistics on this data gave a significant correlation with 0.05 < p < 0.02. Techniques allowing current sample preparation methods to be easily adapted for use with ELISA analysis are also described.
A highly sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the detection of the arylurea herbicides monuron, diuron, and linuron is described. Diuron haptens with two different polymethylene handle locations were evaluated for use as immunizing and/or tracer antigens. One handle, consisting of three or five methylene groups, located at the terminal urea nitrogen distal to the aromatic ring provided the best antibodies and enzyme-labeled haptens with a variety of selectivities. The second methylene handle attachment at the internal urea nitrogen retained the basic structure of the target molecule, but was not recognized by the antibodies produced from the terminal substituted haptens, nor was it useful as an enzyme tracer. Rabbits were immunized with monuron hapten-bovine serum albumin conjugates that contained either a C3 or C5 methylene handle at the terminal nitrogen. The resulting selectivity of the antisera was not related to the immunizing antigen used. The analyses were class or compound selective for the individual herbicides depending upon the antisera used. Widely applied urea herbicides such as diuron, monuron, and linuron demonstrated 50% inhibition values at 0.4,0.5, and 0.8 pg/L and detection limits of 0.04,0.05, and 0.08 pg/L, respectively, in buffer. The test, applied to matrices such as water and orange juice, showed limited matrix effects which could be eliminated by moderate dilution of the sample. The assays were tested for tolerance to methanol, a commonly used solvent in the extraction of diuron from environmental matrices. Up to 50% methanol in the assay had no effect on assay parameters such as the 50% inhibition values. To develop a rational approach for the selection of a hapten for use as an enzyme tracer, a variety of monuron, diuron, and linuron haptens were tested for cross reactivity. Some of these same haptens were then used as hapten enzyme conjugates. In general, haptens recognized about 100 times less well than the "best" hapten were useful as enzyme tracers. In addition, we found that cross reactivity data for esters of carboxylic acid haptens may be better predictors of the binding of the carboxylic acids when conjugated as antigens or enzyme tracers.
The present work describes screening and selection of rabbit antisera raised against the s-triazine herbicides, characterization of antibody cross-reactivity and specificity, and improvements in ELISA sensitivity. These results stem from a comprehensive synthetic approach described previously for the production of s-triazine haptens. Both homologous and heterologous ELISA systems were examined by using heterology based on hapten conjugation position, spacer length, and/or alkyl substitution. Sensitivity to the target analyte and to nonspecific effects was least for homologous systems. Except for extreme differences in spacer length, heterology based on conjugation position provided the largest improvement in sensitivity to the target analyte. Matrix and solvent effects depended on the ELISA system used. Specificity studies using several antibodies in competitive inhibition ELISA with over 30 inhibitors showed that conjugation position and alkyl substitutions were important determinants of antibody specificity and that the antibodies recognized the immunizing hapten better than all other inhibitors tested. The resulting assays were capable of detecting the parent and related s-triazines at low ppb to sub-ppb levels.
The metabolism of the pyrethroid insecticide cypermethrin has been studied in rats using three forms of 14C-labelling (benzyl-, cyclopropyl- and cyano-) and separate cis- and trans- isomers. The proportion of the dose absorbed from the intestines (50–70% at 2–3 mg kg−1) is rapidly metabolised and eliminated. The major reaction is cleavage of the ester bond to afford the constituent cis- and trans- acids which are conjugated with glucuronic acid and eliminated in the urine. The 3-phenoxybenzyl portion of the molecule is probably released as the α-hydroxynitrile, which is converted via the aldehyde into 3-phenoxybenzoic acid. This compound is then largely hydroxylated and eliminated as a sulphate conjugate. The cyanide ion is metabolised via predictable routes, for instance, as thiocyanate. Cypermethrin is hydroxylated to some extent before hydrolysis. Most of this hydroxylation occurs at the methyl group trans to the cyclopropane carboxyl group, and at the 4-position of the phenoxy group. cis- Cypermethrin is slightly more stable than the trans-isomer.
This review of studies on 20 pyrethroids with nine different acid moieties and ten different alcohol moieties reveals a diversity of functional groups undergoing metabolism in mammals, insects, other organisms, and microsomal esterase and oxidase systems. Seventy-nine metabolites are identified from the cis-isomer of permethrin and transpermethrin but fewer from other pyrethroids examined in less detail. The sites and rates of metabolic attack on each pyrethroid depend on the organism or system. Metabolism of pyrethoids by esterase and oxidase action usually limits their toxicity to mammals more than to insects, thereby conferring useful selective toxicity properties.
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of fenpropathrin [(RS)-R-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarbox-ylate]. Polyclonal antisera were isolated from rabbits immunized with two different fenpropathrin hapten conjugates. One hapten contained an amino function; the other contained a carboxyl group for conjugation to carrier proteins. Mollusk hemocyanins, thyroglobulin, and fetuin were used as carrier proteins. The antisera varied greatly in their affinities for fenpropathrin. A homologous assay system using the coating antigen format was the most sensitive. The IC 50 for fenpropathrin was 20 µg/L, and the lower detection limit was 2.5 µg/L. Pyrethroids, such as phenothrin, permethrin, resmethrin, fenvalerate, deltamethrin, cyfluthrin, and cypermethrin, and the pyrethroid metabolites, 3-phenoxybenzoic acid and fenpropathrin acid, did not cross-react significantly in this assay. Ten percent acetone or methanol and a pH of 4 were determined to be optimum assay conditions. Various cationic, anionic, and nonionic detergents had no significant effect on the assay.
Natural pyrethrins from insecticidal pyrethrum extract and pyrethroids (e.g. allethrin, tetramethrin, permethrin, cyphenothrin, cypermethrin, cyfluthrin) are active ingredients in insecticidal formulations such as powder, sprays, impregnated paper for electro-evaporators, mosquito coils, and solutions for wood treatment, all mainly intended for indoor use. Some commercial preparations contain also non-pyrethroid insecticides, such as dichlorovos, propoxur, or phoxim, and piperonyl butoxid as synergist. High-resolution gas-chromatography with oncolumn injection and FID and ECD detection is employed for the analysis of these insecticides in commercial formulations, in air during and after indoor application, and as residues on surfaces. The total input of pyrethroids into a large room amounts to 1 to 30 mg. The concentrations of the pyrethrins and pyrethroids in air (2 to 300 g/m3) and their deposition on surfaces (up to 1000 g/m2) reveal possible exposure of humans by inhalation (e.g. 30 g allethrin or 60 g tetramethrin) or by skin resorption (e.g. 200 g allethrin and up to 1000 g tetramethrin). The insecticides deposited on surfaces and some readily formed transformation products persist for 60 h or longer.
An analytical method for the simultaneous determination of the pyrethroid metabolites 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 samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 g L–1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.
We have developed an enzyme-linked immunosorbent assay (ELISA) for the herbicide simazine with virtually no recognition of propazine and very low (8%) recognition of atrazine. In this research we have developed a generalized “size-exclusion” concept for designing immunogen hapten structures. The hapten should contain appendages smaller than those of the target analyte thereby generating polyclonal antibodies that exclude recognition of analytes larger than the target molecule. The use of this size-exclusion model, when extended to include coating/tracer haptens, is also able to predict suitable structures for ELISA development.
Improved assessments of human exposure to electrophilic chemicals require rapid and inexpensive analytical techniques that can detect specific urinary metabolites at low levels as needed for epidemiological screenings of large populations. The first aim of this study has been to apply rational hapten design strategies to develop a more sensitive and selective enzyme-linked immunosorbent assay for atrazine mercapturic acid. Polyclonal sheep antiserum was generated against an improved hapten, numerous coating antigen chemistries were evaluated, and assay conditions were optimized. An assay was developed with an IC50 of 0.08 +/- 0.02 micrograms/L (K approximately with 10(-)10 M) for atrazine mercapturic acid. The assay exhibited greatest recognition of atrazine mercapturic acid relative to other known urinary metabolites of atrazine as well as other triazine herbicides. The assay was surprisingly selective to atrazine mercapturic acid over the structurally similar simazine mercapturic acid. Urine samples presented matrix effects due in part to the nonspecific effects of urinary salts, but 4-fold dilution of urine achieved an overall method limit of quantitation of 0.3 micrograms/L. Solid-phase extraction strategies were also developed in an attempt to increase the sensitivity of the overall method. However, a weak positive assay response was present in the solid-phase extracts of unspiked urines, resulting in accurate recovery of atrazine mercapturic acid at microgram/L.
A radioimmunoassay for dieldrin and aldrin has been developed which can detect picomole levels of either compound. The specificity of the antibodies, determined with respect to several other organochlorine insecticides, indicates that most of the binding energy is directed to the polychlorinated bicyclic ring system. DDT, 2,4,5-T, dechachlorobiphenyl (a PCB standard), and several other insecticides which may be found in samples containing dieldrin and aldrin should not interfere with the radioimmunoassay.
The mechanisms of action of neuroactive insecticides on the nervous system has been studied for many years. It is now well established that severe neurological symptoms of poisoning with pyrethroids and DDT in mammals and insects are the result of modification of Na+ channel activity. Toshio Narahashi discusses the history, approaches and results of the studies leading to this conclusion. Advanced electrophysiological experiments using voltage clamp and patch clamp, together with ligand-binding and ionic flux experiments, have unveiled unique actions of pyrethroids and DDT of keeping the Na+ channel in the open state for an extremely long period, sometimes as long as several seconds. This modification of Na+ channel properties leads to hyperactivity of the nervous system. These insecticides have also been shown to suppress GABA and glutamate receptor-channel complexes and voltage-activated Ca2+ channels, but the toxicological significance of these actions remains to be seen. The results of these studies provide clues for developing newer insecticides with higher selectivity between mammals and insects and for coping with the problem of insecticide resistance.
1. The pyrethroid insecticide cypermethrin was administered orally to six male volunteers as a single dose of 3.3 mg (cis: trans 1:1) and dermally to six volunteers at a dose of 31 mg/800 cm2 (cis:trans 56:44) as a soya oil-based formulation. Urine samples were collected for up to 5 days and analysed for the metabolites cis and trans 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVA), 3-phenoxybenzoic acid (3PBA) and 3-(4'-hydroxyphenoxy) benzoic acid (4OH3PBA) following an acid hydrolysis procedure. 2. Following oral dosing approx. equal amounts of (cis+trans DCVA) and (3PBA+4OH3PBA) were excreted with peak excretion rates occurring between 8 and 24 h after dosing. The ratio of trans:cis DCVA was on average 2:1. Based on DCVA measurements the amount of cypermethrin absorbed was estimated to be between 27% and 57% (mean 36%) of the administered dose. 3. Peak urinary excretion rates of metabolites occurred between 12 and 36 h after dermal dosing. The amount of metabolites derived from the phenoxybenzyl moiety (3PBA+4OH3PBA) was on average 4 times greater than the amount of (cis+trans DCVA) recovered in urine. The ratio of trans:cis DCVA was, on average 1:1.2. Based on the recovery of the phenoxybenzyl metabolites it is estimated that 0.85-1.8% (mean 1.2%) of the administered cypermethrin was absorbed. 4. These studies demonstrate marked differences in the urinary metabolite profile by the two routes, and provide an improved basis for determining the extent and main route of absorption of cypermethrin under occupational exposure conditions.
1. Most pyrethroids can be divided into two classes on the basis of differences in the signs of toxicity (T- orCS-syndromes). Since these syndromes also correspond to particular aspects of their chemical structure and since the order of appearance of the signs of poisoning is the same in each class, it is concluded that these syndromes originate from a single primary action of the pyrethroid.
2. Pyrethroids cause morphological changes in peripheral nerves of rats when given in high doses. The production of these minor lesions are correlated with a dose of the pyrethroid that causes death in some of the treated rats. Lower doses do not cause these effects. Therefore, the morphological changes are produced as a secondary consequence of the primary action of pyrethroids and are not due to a different form of toxicity.
3. Pyrethroids have been shown to cause functional changes (behavioral) in rats shown as a deficit in performance on an inclined plane. Increases in peripheral nerves of β-glucuronidase and β-galactosidase have also been demonstrated. These increases are a late finding and are considered to be associated with repair processes. As with the morphological changes these enzyme changes are correlated with doses that cause death in some of the animals. There is no firm evidence that the behavioral changes are correlated with either the morphological or biochemcial changes.
4. Available information indicates that all the above changes described in 2 and 3 are reversible or repairable.
5. Many pyrethroids cause an effect in humans termed parasthesia. It seems probable from structure-activity relationships that parathesia, as for their systemic toxicity, is brought about by an action of pyrethroids on sodium channels of the sensory nerves.
6. Studies have been carried out on human volunteers and a guinea pig model has been developed. Subjective human experience and some experimental work indicates that the effects are reversible and result in no permanent change.
7. There is no conclusive evidence that pyrethroids have neurotoxic actions other than those originating from their primary action on sodium channels, through a dissociable interaction with macromolecular component(s).
8. Further research is recommended to extend knowledge of pyrethroids as a class rather than for individual compounds.
9. All available evidence indicates that, at doses of pyrethroids likely to be encountered in working practice, when serious poisoning does not occur, neurotoxicity due to the pyrethroids will not occur except as a reversible transient effect on the skin. This minor warning response is due to the primary action of pyrethroids on sodium channels, a reversible interaction.
This article reviews 573 cases of acute pyrethroid poisoning reported in the Chinese medical literatures during 1983-1988. There were 325 cases of acute deltamethrin poisoning (occupational 158, accidental 167), 196 patients of acute fenvalerate poisoning (occupational 63, accidental 133, including 2 cases of ingestive fenvalerate-organophosphate mixture poisoning), 45 cases of acute cypermethrin poisoning (occupational 6, accidental 39) and 7 cases of other pyrethroid poisoning (occupational 2, accidental 5). The clinical manifestations have been reviewed and analysed. The diagnosis, gradation and differential diagnosis of occupational acute pyrethroid poisoning have been discussed.
1. Dose-excretion studies with cypermethrin (as a 1:1 cis/trans mixture) and alphacypermethrin (one of the two disastereoisomer pairs which constitute cis cypermethrin) were carried out with, in each case, two volunteers per dose level. The studies included (a) single oral alphacypermethrin doses of 0.25 mg, 0.50 mg and 0.75 mg followed by repeated alphacypermethrin doses at the same levels, daily for five days, (b) repeated oral cypermethrin doses of 0.25 mg, 0.75 mg and 1.5 mg daily for five days, and (c) a single dermal application of 25 mg cypermethrin to the forearm. Urine was monitored for the free and conjugated 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid before and after dosing. 2. Metabolism and rate of excretion of a single oral dose of alphacypermethrin was similar to that of cis cypermethrin, on average, 43% of the dose was excreted as the cyclopropanecarboxylic acid in the first 24 h urine. There was no increase in urinary metabolite excretion when alphacypermethrin was administered as a repeated oral dose. Subjects excreted, on average, 49% of the dose as the cyclopropanecarboxylic acid in the subsequent 24 h periods after dosing. 3. There was no increase in the urinary cyclopropanecarboxylic acid excretion when cypermethrin was administered as a repeated oral dose. Subjects excreted, on average, 72% of the trans isomer dose and 45% of the cis isomer dose respectively in the subsequent 24 h periods after dosing. 4. Approximately 0.1% of the applied dermal dose of 25 mg cypermethrin was excreted within 72 h as the urinary cyclopropanecarboxylic acid. No conclusions can be drawn from such urinary excretion data as to the concentration of cypermethrin and its metabolites in the skin or other organs, or the possibility of other routes of metabolism or excretion.
A health survey was conducted on 199 workers engaged in dividing and packaging pyrethroids. The subjects were exposed to fenvalerate at 0.012-0.055 mg/m3 and deltamethrin at 0.005-0.012 mg/m3 in the air with simultaneous skin contact for 0.5-4.5 months. Burning sensations and tightness or numbness on the face appeared in two thirds of the subjects and one third had sniffs and sneezes. Abnormal facial sensations, dizziness, fatigue, and miliary red papules on the skin were more evident in summer than in winter. Neither abnormalities in other organs or systems nor symptoms or signs of acute pyrethroid poisoning were found by interviews, examinations, and laboratory tests. There was no significant difference in plasma levels of NA, cAMP, and cGMP between the examined subjects and the control group. The urine concentration of fenvalerate in the study group ranged from 1.02 to 18.6 micrograms/l; deltamethrin in the urine was present in trace amounts.
Dermal and respiratory exposure to pirimiphosmethyl, dimethoate and permethrin were determined for applicators and operators in greenhouse tomato spraying operations. Dermal exposure is several times higher than the degree of respiratory exposure. Dermal exposure in terms of different parts of the body shows significant differences. For applicators the exposure of hands, arms and legs is the greatest, and the operators are the most exposed on their hands and to a small extent on legs. This fact should be taken into account when providing the workers with suitable protective clothing. The carefully selected technology of spraying also has great significance in decreasing the degree of exposure. Because of the chronic toxicity of dimethoate, all possible methods should be taken to reduce exposure.
1. An analytical method for monitoring human exposure to cypermethrin has been developed, based on the detection of the free and conjugated forms of the urinary metabolite, the cyclopropanecarboxylic acid. 2. Four male subjects were given a single oral dose, ranging from 0.25 mg to 1.5 mg, of a 1:1 cis/trans mixture of cypermethrin, and urine was monitored for the free and conjugated cyclopropanecarboxylic acid. Urinary excretion of the individual metabolites (cis and trans isomers) was similar for the different dosages. Subjects excreted, on average, 78% of the trans isomer dose, and 49% of the cis isomer dose respectively in 24 h. 3. Thus, as in other mammals, ester cleavage and elimination of the cis and trans cyclopropanecarboxylic acid moieties in the free and conjugated form is a major route of metabolism of cypermethrin in man.
The measurement of metabolites constitutes a useful tool for detection of exposure and in pharmacokinetic studies. Epoxidation with subsequent glutathione conjugation and mercapturic acid formation is an important deactivation pathway for naphthalene, a toxin which presumably causes lung disease. The mercapturic acid conjugates of naphthalene [NaphMA (1), N-acetyl-S-(1,2-dihydro-1-hydroxy-2-naphthyl)cysteine (1a), and N-acetyl-S-(1,2-dihydro-2-hydroxy-1-naphthyl)cysteine (1b)], its most important urinary metabolites, and other structurally related derivatives, such as N-acetyl-S-(1,2,3,4-tetrahydro-2-hydroxy-1-naphthyl) cysteine (2), N-acetyl-S-(3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl)cysteine (3), and N-acetyl-S-(2-hydroxy-1-phenylethyl)cysteine (4a) and N-acetyl-S-(2-hydroxy-2-phenylethyl)cysteine (4b) as an isomeric mixture, were synthesized to develop an ELISA (enzyme-linked immunosorbent assay) for the specific detection of NaphMA (1). Compound 1, as an isomeric mixture, was used to raise antibodies by immunizing six rabbits with the corresponding KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) derivatives (1KLH and 1BSA). The remaining compounds were covalently attached to BSA, conalbumin, and ovalbumin to be used as coating antigens. The best assay was obtained in a homologous system combining serum Ab2357 (1KLH) and 1BSA as coating antigen. The immunoassay has an I50 of 4-6 ng/mL and a detection limit of 1-2 ng/mL. Because of the known instability of the mercapturic acid conjugate of naphthalene 1, leading to the fully aromatic compound 20, a system involving HPLC is described to check the stability of the NaphMA stock solutions used in the assay. Cross-reactivity studies show high specificity toward the NaphMA. Other related compounds as well as the dehydrated derivative 20 are not recognized by the antibody in this ELISA system.
Enzyme-linked immunosorbent assays (ELISAs) are reported for the detection of atrazine and its principle metabolite in human urine. The ELISAs can be used with crude urine or following extraction and partial purification by methods described in this report. GC, MS, and HPLC techniques were used to confirm and complement the ELISA methods for qualitative and quantitative detection of urinary metabolites. A series of samples from workers applying this herbicide confirmed a mercapturic acid conjugate of atrazine as a major urinary metabolite. The mercapturate was found in concentrations at least 10 times that of any of the N-dealkylated products or the parent compound. Atrazine mercapturic acid was isolated from urine using affinity extraction based upon a polyclonal antibody for hydroxy-s-triazines and yielded products sufficiently pure for structure confirmation by MS/MS. In a pilot study monitoring applicators, a relationship between cumulative dermal and inhalation exposure and total amount of atrazine equivalents excreted over a 10-day period was observed. On the basis of these data, we propose that an ELISA for the mercapturate of atrazine could be developed as a useful marker of exposure.
The extensive worldwide efforts of structural modification of natural pyrethrins for better performances have resulted in successful development of a wide variety of synthetic pyrethroids with tremendously high efficacy, knock-down activity or vapor action, and/or with acceptable environmental stability and safety. Currently these pyrethroids including their preferentially manufactured stereoisomers are widely used in agriculture, and for public health as well as household insect control. The detailed toxicology and metabolism studies intended to attain human risk assessment have revealed that with voltage-dependent sodium channel as target site pyrethroids induce pronounced repetitive activity characterized grossly by tremor, hypersensitivity, choleoathetosis, and salivation. In addition, so-called cyano-pyrethroids cause transient skin paresthesia in workers. With regard to tumorigenicity, mutagenicity, teratogenicity and developmental toxicity, no significant findings have been reported. Pyrethroids are eliminated from the animals quite rapidly and completely, undergoing oxidation and ester hydrolysis followed by various conjugations, with low tissue residues. Thus, overall, sound scientific bases exist for human risk assessment under the present usage conditions.
The objective of this study was to perform biological monitoring of subjects who are occupationally exposed to pyrethroids. The study group consisted of 30 pest control operators exposed to cyfluthrin, cypermethrin or permethrin. After exposure, 24-h urine samples were collected and 20 ml of blood was drawn. The pyrethroid metabolites cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzoic acid and fluorophenoxybenzoic acid were determined in the urine samples (limit of detection: 0.5 micrograms/l) by GC MS and the pyrethroids in plasma (limit of detection: 5 micrograms) GC-ECD. The concentrations of metabolites in the urine of the pest control operators ranged between < 0.5 micrograms/l and 277 micrograms/l urine. The concentrations of cyfluthrin, cypermethrin and permethrin in the plasma were below the limits of detection (< 5 micrograms/l). To test if the metabolites are specific for pyrethroid exposure, they were determined in the urine of non-exposed subjects (n = 40). In no case could pyrethroid metabolites be detected. A cyfluthrin elimination experiment showed that cyfluthrin metabolites are eliminated following first-order kinetics (t 1/2 = 6.4 h). Storage experiments demonstrate that frozen urine samples (-21 degrees C) show no significant losses of metabolites within a year. In contrast, pyrethroids stored in plasma are susceptible to further biodegeneration.
The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, fenvalerate, phenothrin and beta-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 microg 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 microg g(-1) creatinine.
1. Nine male volunteers were exposed to the pyrethroid insecticide cyfluthrin. The study was performed in an exposure room, where an aerosol containing cyfluthrin was sprayed to obtain atmospheres with mean cyfluthrin concentrations of 160 and 40 micrograms/m3. Four volunteers were exposed for 10, 30 and 60 min at 160 micrograms/m3 and another five volunteers were exposed for 60 min at 40 micrograms/m3. For 160 micrograms/m3 exposure urine samples were collected before and immediately after exposure as well as for the periods 1-2, 2-3, 3-4, 4-5, 5-6, 6-12 and 12-24 h after exposure. For 40 micrograms/m3 exposure urine samples were collected before and 2 h after exposure. 2. The main urinary cyfluthrin metabolites, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropane carboxylic acid (DCCA) and 4-fluoro-3-phenoxybenzoic acid (FPBA), were determined. The limit of detection (LOD) for all metabolites was 0.0025 microgram in an urine sample of 5 ml (0.5 microgram/l). After inhalative exposure of 40 micrograms cyfluthrin/m3 air for 60 min, the amount of metabolites in urine collected in the first 2 h after exposure was less than the LOD, namely 0.14 microgram for cis-DCCA, 0.15-0.28 microgram for trans-DCCA and 0.12-0.23 microgram for FPBA. 3. Of the metabolites, 93% was excreted within the first 24 h (peak excretion rates between 0.5 and 3 h) after inhalative exposure of 160 micrograms/m3. The mean half-lives were 6.9 h for cis-DCCA, 6.2 h for trans-DCCA and 5.3 h for FPBA. 4. The mean trans-:cis-DCCA ratio was 1.9 for the time course as well as for each subject. 5. The amount of metabolites in urine depends on the applied dose, on the exposure time and shows interindividual differences.
A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as immunogens. Five antisera were produced and screened against eight different coating antigens. The assay that had the least interference and was the most sensitive for esfenvalerate was optimized and characterized. The I(50) for esfenvalerate was 30 +/- 6.2 microg/L, and the lower detection limit (LDL) was 3.0 +/- 1.8 microg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction (SPE) was used for water matrix. With this SPE step, the LDL of the overall method for esfenvalerate was 0.1 microg/L in water samples.
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