Activation of Phosphorothionate Pesticides Based on a Cytochrome

Zheng Han , Chensen Chi , Bing Bai , Gang Liu , Qinxiong Rao , Shaojie Peng , Hong Liu , Zhihui Zhao , Dabing Zhang , Aibo Wu. Analytica Chimica Acta ...
0 downloads 0 Views 114KB Size
Download PDF
Anal. Chem. 2004, 76, 1720-1725

Activation of Phosphorothionate Pesticides Based on a Cytochrome P450 BM-3 (CYP102 A1) Mutant for Expanded Neurotoxin Detection in Food Using Acetylcholinesterase Biosensors Holger Schulze, Rolf D. Schmid, and Till T. Bachmann*

Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors. Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form. In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms. Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis. In contrast, a genetically engineered triple mutant of P450 ΒΜ-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements. The wild-type protein was unable to do so. In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon. The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 µg/kg within an overall assay time of 95 min. Organophosphorus pesticides are the most common pesticides involved in food poisoning1 and an issue for consumer safety. They are therefore important target molecules in the search for fast and sensitive analytical methods. Acetylcholinesterase (AChE) inhibition tests have been shown to be suitable for insecticide detection.2-4 AChE is inhibited by the two most important groups of insecticides, namely, organophosphates and carbamates.5 Out of the organophosphate insecticides applied worldwide, most belong to the group of phosphorothionates (e.g., ∼80% in Germany6). Phosphorothionates usually exhibit a considerably reduced inhibitory effect toward AChE and therefore pose an * E-mail: [email protected]. (1) Karalliedde, L.; Senanayake, N. eJIFCC [online computer file] 1999, 11. (2) Schulze, H.; Scherbaum, E.; Anastassiades, M.; Vorlova, S.; Schmid, R. D.; Bachmann, T. T. Biosens. Bioelectron. 2002, 17, 1095-105. (3) Skladal, P. Food Technol. Biotechnol. 1996, 34, 43-9. (4) Schulze, H.; Vorlova, S.; Villatte, F.; Bachmann, T. T.; Schmid, R. D. Biosens. Bioelectron. 2003, 18, 201-9. (5) Fukuto, T. R. Environ. Health Perspect. 1990, 87, 245-54.

1720 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

analytical challenge, which is addressed in this study. AChE biosensors have some advantages over standard methods for insecticide detection that are based on gas chromatography (GC) or high-performance liquid chromatography (HPLC) coupled with mass selective detectors.7,8 The former tests are much faster and need less sophisticated and expensive instruments and well-trained personal. GC/MS and HPLC-MS methods are restricted to a limited analyte spectrum. Complex food matrixes such as citrus fruits for example require special multiresidue methods.9 The application of AChE biosensors in the analysis of food leads to a substantially reduced risk of false negatives of these especially hazardous insecticides.10 However, a particular disadvantage of AChE biosensors is the use of only one AChE enzyme, which does not provide specific information about a particular analyte but a sum parameter of AChE inhibition. More detailed qualitative and quantitative information about targeted compounds can for example be obtained when using a multisensor with AChEs of different origin or genetically engineered mutants that is combined with data processing based on artificial neural networks.11,12 The present study examines an AChE biosensor that is based on the enzymatic cleavage of acetylthiocholine. The resulting thiocholine is subsequently anodically oxidized on screen-printed disposable thick-film electrodes, which generates a current that is proportional to the AChE activity. Unfortunately, nonmetabolized organophosphates with a PdS moiety, e.g., the phosphorothionates, show very low inhibition of AChE due to the lower reactivity of the PdS group caused by the minor electronegativity of sulfur compared to oxygen.5 However, AChE inhibition is greatly increased in vivo. The observed toxic effect of phosphorothionates represents the sum of the competitive biochemical processes of activation and detoxification.13 With regard to the analysis of water, there is a standardized method to activate phosphorothionates that is based on a N-bromosuccinimide (NBS) (6) Roth, M.; Weisser, W. Jahresbericht des Chemischen und Veterina ¨ runtersuchungsamtes Stuttgart, CVUA Stuttgart: Fellbach, Germany, 2000. (7) Martinez, C. R.; Gonzales, R. E.; Moran, A. M. J.; Mendez, H. J. J. Chromatogr. 1992, 607, 37-45. (8) Pylypiw, H. M. J. AOAC Int. 1993, 76, 1369-73. (9) Anastassiades, M.; Scherbaum, E. Dtsch. Lebensm.-Rundsch. 1997, 10, 31627. (10) Kwong, T. C. Ther. Drug Monit. 2002, 24, 144-9. (11) Bachmann, T. T.; Schmid, R. D. Anal. Chim. Acta 1999, 401, 95-103. (12) Bachmann, T. T.; Leca, B.; Villatte, F.; Marty, J.-L.; Fournier, D.; Schmid, R. D. Biosens. Bioelectron. 2000, 15, 193-201. (13) Jokanovic, M. Toxicology 2001, 166, 139-60. 10.1021/ac035218t CCC: $27.50

© 2004 American Chemical Society Published on Web 02/19/2004

reaction.14 There are several chemical oxidation procedures with bromine15,16 or chlorine, which is generated in a hydrolysis reaction of sodium chloride.17,18 Here we describe a new enzymatic activation method for phosphorothionates that allows their detection by AChE biosensors in various matrixes including food. The method is based on a procaryotic cytochrome P450 and was developed in accordance with in vivo biotransformation of phosphorothionates, which are catalyzed by microsomal monooxygenases such as the human cytochrome P450 isoforms 3A3 and 2D6.19 Cytochrome P450 ΒΜ-3 is a soluble P450 enzyme from Bacillus megaterium.20,21 It is catalytically self-sufficient and contains both monooxygenase and reductase domains on a single polypeptide chain. The assumed natural function of the enzyme is the subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. NADPH is used as external electron donor.22 A triple mutant of cytochrome P450 ΒΜ-323 was used for the activation of phosphorothionates. We have investigated the potential of the wild-type (WT) and mutant P450 ΒΜ-3 for their use in the biotransformation of phosphorothionates by analyzing the reaction products with GC/MS/MS. The transformation efficiency and the applicability of the method for food analysis were tested. EXPERIMENTAL SECTION Reagents. All chemicals were of analytical reagent grade or higher quality and were purchased from Fluka (Neu-Ulm, Germany) or Sigma-Aldrich (Deisenhofen, Germany). NADPH was obtained from Ju¨lich Fine Chemicals (Ju¨lich, Germany). Pesticide standards and Tween 20 were purchased from Riedel-de Hae¨n (Seelze, Germany). Insecticide stock solutions were prepared in ethanol. AChE (EC 3.1.1.7) from electric eel (type V-S, 970 U/mg) was obtained from Sigma-Aldrich. Escherichia coli strain DH5R (supE44, lacU169 (80lacZ M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) from Clontech (Heidelberg, Germany) was used for protein expression of WT and mutant cytochrome P450 ΒΜ-3. HIPP (Pfaffenhofen, Germany) apple pap infant food (Baby-Apfel, apple with rice), obtained from a local store, was used as test matrix for the analysis of food. Large-Scale Production of Wild-Type (P450 BM-3 WT) and Mutant P450 BM-3. As described previously,24 P450 ΒΜ-3 WT and a triple mutant (Phe87Val, Leu188Gln, Ala74Gly)23 were expressed in the E. coli strain DH5R under the control of the strong temperature-inducible PRPL promoter of pCYTEXP1. A fermenter (type LP351; Bioengineering, Wald, Switzerland) containing 30 L of Luria-Bertani medium supplemented with 100 µg/ (14) DIN38415-1 Dtsch. Norm 1995. (15) Young, K. A.; Lee, H. S.; Park, Y. C.; Lee, Y. T. Environ. Res. 2000, 84, 303-9. (16) Kumaran, S.; Tran-Minh, C. Anal. Biochem. 1992, 200, 187-94. (17) Evtugyn, G. A.; Rizaeva, E. P.; Stoikova, E. E.; Latipova, V. Z.; Budnikov, H. C. Electroanalysis 1997, 9, 1124-8. (18) Ivanov, A. N.; Evtugyn, G. A.; Lukachova, Lilia V.; Karyakina, Elena E.; Budnikov, H. C.; Kiseleva, S. G.; Orlov, A. V.; Karpacheva, G. P.; Karyakin, A. A. IEEE Sens. J. 2003, 3, 333-40. (19) Sams, C.; Mason, H. J.; Rawbone, R. Toxicol. Lett. 2000, 116, 217-21. (20) Narhi, L. O.; Fulco, A. J. J. Biol. Chem. 1987, 262, 6683-90. (21) Narhi, L. O.; Fulco, A. J. J. Biol. Chem. 1986, 261, 7160-9. (22) Boddupalli, S. S.; Estabrook, R. W.; Peterson, J. A. J. Biol. Chem. 1990, 265, 4233-9. (23) Li, Q. S.; Schwaneberg, U.; Fischer, P.; Schmid, R. D. Chem.-Eur. J. 2000, 6, 1531-6. (24) Schwaneberg, U.; Schmidt-Dannert, C.; Schmitt, J.; Schmid, R. D. Anal. Biochem. 1999, 269, 359-66.

mL ampicillin was inoculated with 800 mL of overnight culture (OD600 ) 0.8-1.0). The fermentation was carried out with an aeration of 25 L/min and a stirrer speed of 400 rpm at 37 °C. P450 expression was induced by a temperature shift from 37 to 42 °C for 5 h. After induction, the amount of dissolved oxygen was lowered by reducing aeration to 5.7 L/min and stirrer speed to 150 rpm. E. coli cells containing the P450 ΒΜ-3 were harvested by cross-flow filtration using a Millipore stainless steel holder (Eschborn, Germany) with a Filtron (Dreieich, Germany) Centrasette Omega (0.3 µm) membrane followed by centrifugation of the 15-fold concentrated suspension at 9200g for 20 min. Preparation of E. coli DH5r Cell Extracts. Up to 15 g (wet weight) of DH5R in 20 mL of potassium phosphate buffer ((PBS) 50 mM, pH 7.5) was thawed on ice. After sonifying the cells four times for 1 min in an ice bath (Branson Sonifier W250, Dietzenbach, Germany; output level 80 W, duty cycle 20%), the suspension was centrifuged for 30 min at 32500g. The supernatant (crude extract) was stored at -20 °C before using it for the activation of phosphorothionates. P450 ΒΜ-3 concentrations were measured by the CO differential spectra method as described by Omura and Sato,25 using an extinction coefficient of  ) 91 mM-1 cm-1 with a scan rate of 125 nm/min at 0.1-nm intervals. The concentration was calculated from the difference in absorption between the carbonyl complex of the ferrous form (at 450 nm) and the ferrous form (at 490 nm). A 250-µL aliquot of the cell extract containing the P450 ΒΜ-3 WT or mutant was 10 times diluted with potassium phosphate buffer (50 mM, pH 7.5). After the addition of a catalytic amount of sodium dithionite, the reaction volume (2.5 mL) was divided into 2 parts. CO gas was added into one solution for 1 min. The other part was used as reference solution for the spectrophotometric measurement, which was carried out between 400 and 500 nm. Chemical Oxidation of Phosphorothionates in Buffer. A 100-µL NBS solution (0.4 g/L in water) was added to 9.8 mL of sample solution (final NBS concentration, 4 mg/L) and mixed in an ultrasonic bath for 10 min. Subsequently, 100 µL of ascorbic acid solution (4 g/L in water) was added to remove excessive NBS by mixing in an ultrasonic bath for 10 min. Chemical Oxidation in Food. Ten grams of the food sample was mixed with 7.8 mL of PBS (1 M, pH 7,5) and 2 mL of NBS solution (2 g/L yielding a final concentration of 200 mg/L). After 5 min of stirring and 10 min of incubation in an ultrasonic bath, 200 µL of ascorbic acid solution (200 g/L) was added following again 5 min of stirring and 10 min of incubation in an ultrasonic bath. The oxidation reaction was performed with varying NBS concentrations followed by the addition of a 10-fold concentration of ascorbic acid. GC/MS/MS Analysis. Parathion or chlorpyrifos was dissolved in PBS (50 mM, pH 7.5) (final concentration, 4000 µg/L) and preincubated with 0.32 µM P450 ΒΜ-3 WT or mutant for 6 min. Activation was started by adding 1 mM NADPH (aqueous solution). After 1 h, the organophosphates (final volume, 4 mL) were extracted with 2 mL of dichloromethane. After 30 min of centrifugation, the aqueous phase was extracted again with 2 mL of dichloromethane after 1 mL of saturated NaCl solution was added to the aqueous phase. The combined dichloromethane phase was dried over sodium sulfate. (25) Omura, T. I.; Sato, R. J. J. Biol. Chem. 1964, 94, 2370-8.

Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

1721

The analysis was performed with a Star 3400CX gas chromatograph (Varian, Palo Alto, CA) coupled with an Iontrap Saturn 4D GC/MS/MS (Varian) with a EI of 70 eV. The following oven program was used: 45 °C for 1 min, then 20 °C/min to 200 °C, followed by 7 °C/min to 300 °C, and finally 300 °C for 10 min. P450 BM-3 Pretreatment of Aqueous Samples. Phosphorothionate solutions of different concentrations diluted with phosphate buffer (50 mM, pH 7.5 for aqueous samples and 1 M, pH 7.5, for food samples) were preincubated with 0.32 µM P450 ΒΜ-3 WT or mutant (as crude extract) for 6 min. Activation was started by adding 1 mM NADPH (aqueous solution). After 40 min, this solution (final volume, 0.4 mL) was used as incubation solution for the optical AChE activity assay. P450 BM-3 Pretreatment of Food Samples. From food samples that were spiked with phosphorothionates in the desired concentration, 1.6 g was mixed with phosphate buffer (1 M, pH 7.5) and 0.32 µM P450 ΒΜ-3 WT or mutant (as crude extract). After 6 min of preincubation, 1 mM NADPH (0.8 mL of aqueous solution) was added to a final sample amount of 4 g. After 40 min, this solution was used as incubation solution for the electrochemical AChE biosensor test. During the incubation time, the solution was mixed with an overhead stirrer. Optical AChE Activity Assay. The AChE activity was determined according to the method of Ellman et al.26 with minor modifications. A 100-µL aliquot of the sample solution (e.g., activated phosphorothionate solution) was combined with 690 µL of PBS (50 mM, pH 7.5), 100 µL of 5,5′-dithiobis(2-nitrobenzoic acid) solution (7.8 mM in PBS), and 100 µL of AChE solution in a 1-mL cuvette. After 30 min of incubation, the residual AChE activity was measured by adding 10 µL of acetylthiocholine iodide (100 mM aqueous solution) against a blank consisting of the same mixture without AChE. The percentage of AChE inhibition was calculated from the difference between original and residual activity, divided by the original activity. The original AChE activity was determined according to the method described above but without the addition of the incubation solution. In this case, 790 µL of PBS was used for the test. AChE Biosensor Measurement. AChE biosensors were produced as described earlier11 by screen printing using a DEK 249 screen printer (DEK Ltd., Weymouth, England). They consist of an Ag/AgCl reference electrode and a 7,7,8,8-tetracyanoquinodiemthane-graphite working electrode. Electric eel AChE was immobilized on top of the working electrodes by cross-linking in glutaraldehyde vapor. All sensor experiments to determine AChE activity were carried out in a stirred buffer solution (10 mM PBS, 50 mM NaCl, pH 7.5) at room temperature. The enzyme activity was determined by monitoring thiocholine formed by enzymatic hydrolysis of acetylthiocholine chloride (final concentration, 1 mM). Thiocholine was detected by oxidation at +100 mV versus Ag/AgCl. For use in inhibition experiments, the biosensor was incubated in the above-described food sample for 30 min at room temperature. Before each measurement of the activity of the immobilized enzyme, the electrodes were placed in PBS containing 1% Tween 20 for 15 min and then washed with pure PBS.2 (26) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88-92.

1722 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

Figure 1. AChE inhibition caused by different organophosphate concentrations in phosphate buffer using the optical AChE activity assay. Chlorpyrifos and parathion were activated either with the P450 ΒΜ-3 mutant or chemically by NBS. Also depicted for comparison is the AChE inhibition caused by equal paraoxon concentrations and by nonmetabolized parathion.

RESULTS AND DISCUSSION Direct Detection of Phosphorothionates by AChE Inhibition. Metabolized phosphorothionates are generally very strong in vivo inhibitors of AChE. However, they show only a marginal inhibitory effect in their pure nonmetabolized form. Accordingly, nonactivated parathion did virtually not lead to any inhibition of AChE. A concentration of 100 µg/L parathion caused only a marginal AChE inhibition of 12% (Figure 1). And even a much higher parathion concentration of 2000 µg/L only led to 19% AChE inhibition. These results confirm former observations.13,27 Barber et al., for example, could not detect any AChE inhibition after 60min incubation with 291 µg/L parathion at 37°C and only 10% inhibition when 2910 µg/L parathion was used.27 Obviously, it is necessary to transform phosphorothionates into their corresponding oxo forms before they can be reliably detected with AChE assays. Such assays must span a concentration range of 10-10 000 µg/kg to comply with for the maximum residue limits in food set up by EU regulations.28 Chemical Oxidation of Phosphorothionates in Food Samples. Initially, we attempted to activate the pesticides by a standard procedure, i.e., chemical oxidation with NBS.14 However, this approach was unsuitable for the analysis of food (Table 1). Even a NBS concentration of 20 mg/L did not lead to an inhibition of the AChE biosensor after incubation in apple pap infant food spiked with 20 µg/kg parathion. This is a 5 times higher concentration than usually applied in aqueous solutions. When the same concentration of NBS was used in an aqueous parathion solution, an AChE biosensor inhibition of 42% was observed. The control without any parathion caused a 10% AChE inhibition already, indicating that 20 mg/L NBS is a too high concentration for aqueous phosphorothionate samples. A control with 20 µg/ kg paraoxon led to an AChE inhibition of 40%. One might thus assume that ascorbic acid, which is a widespread food additive or natural ingredient, impedes chemical oxidation with NBS in food samples but not in water. According to the supplier, the investigated food sample also contained ascorbic acid. Another reason for the poor activation of phosphorothionates in food (27) Barber, D.; Correll, L.; Ehrich, M. Toxicol. Sci. 1999, 47, 16-22. (28) EC Official J. Eur. Communities 2000, L244, 76.

Figure 2. GC/MS/MS spectra of the reaction products of the conversion of parathion (MW ) 291) to paraoxon (MW ) 275) by the cytochrome P450 ΒΜ-3 mutant (right). The left spectrum shows the product of the reaction with WT P450 ΒΜ-3. In this case, only parathion could be detected. Table 1. AChE Inhibition of Apple Pap Infant Food and Water Spiked with 20 µg/kg Parathion after Chemical Activation with Different NBS Concentrationsa NBS (mg/L)

apple pap

20 200 1600

0 9 25

AChE inhibition (%) control water 0 nab 23

42 na na

control 10 na na

a Controls contained NBS but no parathion. b na, not analyzed; amperometric measurement with immobilized AChE.

samples by NBS might be the presence of various other components that might also react with the nondiscriminating oxidation agent. If so, high NBS concentrations could solve this problem. However, even a NBS concentration of 1.6 g/L led to only a reduction of AChE activity of 25 and 23%, respectively, after incubation in food with and without parathion. Consequently, it is necessary to find alternative activation methods for phosphorothionates in food samples in order to make AChE inhibition tests applicable. GC/MS/MS Analysis of the Reaction Products of P450Catalyzed Activation. Cytochrome P450 ΒΜ-3 was chosen to examine other, more robust and specific ways to transform thionates into their corresponding AChE inhibiting oxo forms. The transformation efficiency of the wild-type enzyme and a triple mutant (Phe87Val, Leu188Gln, Ala74Gly) was investigated. The products of the transformation reaction of parathion and chlorpyrifos at concentrations of 4 g/L buffer were analyzed with GC/ MS/MS. This high concentration was necessary because of the low sensitivity of the GC/MS/MS compared to the AChE assay. Figure 2 shows that the mutant yielded a quasi-quantitative transformation of parathion (retention time, 16.82 min) to paraoxon (16.15 min) whereas the reaction with the wild-type cytochrome P450 ΒΜ-3 did not produce any paraoxon. The assignment was confirmed by the analysis of pure parathion (MW ) 291) and paraoxon (MW ) 275). The ratio of the peak areas of paraoxon/ parathion of the mutant transformation was 15:1. Taking into account the different signal intensities of paraoxon and parathion, it can be concluded that the cytochrome P450 ΒΜ-3 mutant yielded a transformation rate of 98.9% (parathion gives a 6 times

higher signal than paraoxon). Tests with chlorpyrifos gave similar results (data not shown). The P450 ΒΜ-3 WT did not catalyze any chlorpyrifos oxon formation. The reaction of chlorpyrifos with the triple mutant yielded a quasi-quantitative transformation into chlorpyrifos oxon. Obviously, the introduction of three mutations (Phe87Val, Leu188Gln, Ala74Gly) led to a mutant that is able to activate phosphorothionates. Originally, this mutant was produced for use in the biocatalysis of industrially interesting fine chemicals and was shown to be able to turn indole into indigo.23 The substitution of a bulky phenylalanine for an alanine at position 87 of the enzyme (F87A) led to a larger binding pocket.29 As the structure of the palmitoleic acid/P450 ΒΜ-3 heme domain complex shows, the Leu188 site is brought into direct contact with the substrate upon repositioning of the Phe/Gly loop. Therefore, the heme domain probably plays an important role in either binding or orienting the substrate.30 It can be presumed that the three mutations increase the size of the active site and thus make it accessible for bigger and unusual substrates such as phosphorothionates. AChE Inhibition Test with Enzymatically Activated Phosphorothionates. The activation of phosphorothionates in aqueous solutions by the P450 ΒΜ-3 mutant was investigated with a subsequent optical AChE assay according to Ellman et al.26 Chlorpyrifos, chlorpyrifos methyl, methidathion, and parathion were chosen because they are often found in fruit and vegetables. They differ substantially in their chemical structure and in the size of the alkoxy moiety (Figure 3). Chlorpyrifos was frequently detected (33%) in a fruit monitoring program carried out by a German state food authority in Stuttgart (Chemical and Veterinary Official Laboratory (CVUA) Stuttgart, Germany).6 During this monitoring program, carried out in the year 2000, 91 domestic and 357 imported fruit samples were analyzed. Only the fungicide carbendazim was found more frequently (39%). Other insecticides such as methidathion, chlorpyrifos methyl, and parathion were detected in 11, 6, and 1% of the analyzed fruits, respectively. The results of the AChE assay are depicted in Figures 1 and 4. Phosphorothionate concentrations in the range between 1 and 100 µg/kg led to inhibition of AChE after activation with the P450 (29) Oliver, C. F.; Modi, S.; Sutcliffe, M. J.; Primrose, W. U.; Lian, L. Y.; Roberts, G. C. Biochemistry 1997, 36, 1567-72. (30) Li, H.; Poulos, T. L. Nat. Struct. Biol. 1997, 4, 140-6.

Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

1723

Figure 3. Chemical structures of organophosphates analyzed in this study.

Figure 4. AChE inhibition caused by different chlorpyrifos methyl and methidathion concentrations in phosphate buffer using the optical AChE activity assay after activation with the P450 ΒΜ-3 mutant.

ΒΜ-3 mutant. The activation efficiency of NBS oxidation of chlorpyrifos and parathion was determined for comparison as well as the AChE inhibition caused by paraoxon, the oxo variant of parathion (Figure 1). In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS. However, neither activation method yielded an AChE inhibition that was as high as with paraoxon. Barber et al. compared chemical oxidation of phosphorothionates using bromine solution with the activation using liver microsomes (RLM), which were isolated from male rats.27 The AChE activity was determined in treated human SH-SY5Y neuroplastoma cells, and a dose-dependent AChE inhibition was observed. However, the transformation efficiency was much lower than with the P450 ΒΜ-3 mutant. Barber et al. obtained less than 50% AChE inhibition for parathion and chlorpyrifos. The incubation of parathion and chlorpyrifos with RLM resulted in multiple metabolites, and it is likely that other detoxification mechanisms reduced the activation efficiency of phosphorothionates by RLM. The high transformation efficiency obtained with the P450 ΒΜ-3 mutant hints to a lack of detoxification pathways, which increased the amount of AChE inhibiting substances. An alternative method for the activation of organophosphorus pesticides was described by Hernandez and co-workers in which chloroperoxidase from Caldariomyces fumago was used instead of cytochrome P450.31 In contrast to microsomal preparations of P450s from rat liver, no hydrolysis products of the detoxification mechanisms were detected after enzymatic oxidation. The specific activity (expressed (31) Hernandez, J.; Robledo, N. R.; Velasco, L.; Quintero, R.; Pickard, M. A.; Vazquez-Duhalt, R. Pestic. Biochem. Physiol. 1998, 61, 87-94.

1724

Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

in moles of phosphorothionates oxidized per mole of biocatalyst per unit of time (s-1)) varied from 2.4 for phosmet to 3.4 for chlorpyrifos and 5.6 for parathion. The transformation efficiency with regard to AChE inhibition was not determined, and thus, no information is available about the potential applicability of chloroperoxidase-mediated activation of phosphorothionates for subsequent use in AChE biosensors. The majority of in vitro studies on the enzymatic activation of phosphorothionates have been carried out with hepatic microsomal preparations or with purified cytochrome P450 isoforms.19,32,33 However, these studies focus exclusively on an understanding of the biotransformation mechanism of these compounds but had no practical application in mind. Recombinant enzymes have several advantages over native P450 isoforms. P450 ΒΜ-3 can be produced in large quantities, using recombinant E. coli expression systems in a fermentation scale and no mammals need to be killed. Furthermore, the handling of E. coli is much easier and less costly than that of rats, for example. The protein structure and thus its function can be altered by genetic engineering. The prokaryotic P450 ΒΜ-3 has additional advantages over the mammalian monooxygenases; it is far more stable than most other P450s and has the highest catalytic activity of them all.22,34-37 P450 ΒΜ-3 contains a cytochrome P450 domain and a flavoprotein NADPH-cytochrome P450 reductase domain in a single polypeptide chain whereas most other P450s require an additional flavoprotein reductase, which renders them unsuitable for biosensor applications. In contrast to the membrane-associated microsomal and mitochondrial P450s, bacterial P450s are usually water soluble.38 Activation of Phosphorothionates in Food Samples. The applicability of the novel enzymatic activation method for the analysis of food samples was examined. For this purpose, P450 ΒΜ-3 activation was implemented into a recently developed biosensor test for neurotoxic compounds in food.2 The principle of the biosensor and the combination of P450 activation of phosphorothionates with the AChE assay is illustrated in Figure 5. The P450 ΒΜ-3 mutant activation of phosphorothionates was (32) Levi, P. E.; Hodgson, E. Toxicol. Lett. 1985, 24, 221-8. (33) Hodgson, E.; Rose, R. L.; Ryu, D. Y.; Falls, G.; Blake, B. L.; Levi, P. E. Toxicol. Lett. 1995, 82-83, 73-81. (34) Guengerich, F. P. J. Biol. Chem. 1991, 266, 10019-22. (35) Munro, A. W.; Leys, D. G.; Mclean, K. J.; Marshall, K. R.; Ost, T. W. B.; Daff, S.; Miles, C. S.; Chapman, S. K.; Lysek, D. A.; Moser, C. C.; Page, C. C.; Dutton, P. L. Trends Biochem. Sci. 2002, 27, 250-7. (36) Roberts, G. C. Chem. Biol. 1999, 6, R269-72. (37) Schwaneberg, U.; Appel, D.; Schmitt, J.; Schmid, R. D. J. Biotechnol. 2000, 84, 249-57. (38) Fulco, A. J. Annu. Rev. Pharmacol. Toxicol. 1991, 31, 177-203.

Figure 5. Principles of the biosensor test for the detection of phosphorothionates. The required time for each step is indicated. Initial and remaining AChE activities were recorded amperometrically.

Figure 6. AChE inhibition caused by different organophosphate concentrations in food samples after activation with the P450 ΒΜ-3 mutant using amperometric measurement with immobilized AChE. AChE inhibition caused by paraoxon in buffer is depicted for comparison.

carried out in solution in the neutralized food sample. The molarity of the buffer (1 M PBS) that was used for the neutralization of the sample obviously did not disturb the activity of the enzyme; the transformation efficiency of parathion was as high in food samples as in buffer (Figure 6). In contrast to aqueous solutions, the transformation efficiency of chlorpyrifos in food is slightly lower; AChE inhibition reached on average 68% of the values obtained in buffer solutions. CONCLUSION The new enzymatic method for activation of phosphorothionates along with an AChE inhibition assay increases the spectrum of analytes that can be detected by biosensors. We present the

first biosensor method that has the potential capability of screening food for all particularly hazardous carbamate and organophosphate pesticides. All previously published AChE assays that target insecticides in food samples were restricted to carbamates and the very rare organophosphates that are applied directly as oxo variants.39-42 Our screening test could substantially increase consumer safety. The system provides information about the toxicity of a sample since the sensor detects the sum of all neurotoxic compounds that are present in the food. This is a big advantage over standard GC/MS or LC-MS methods, which have a restricted analyte spectrum, depending on the multiresidue method applied. The new biosensor test reduces substantially the risk of obtaining false negative results. In addition, the analysis is around eight times faster than GC/MS tests. One test takes only 95 min whereas GC/MS measurements require approximately one working day. Furthermore, the biosensor test is very sensitive and thus suitable for the monitoring of infant food. Samples were analyzed in cooperation with the CVUA Stuttgart (Germany), and amounts as low as 10 µg/kg could reliably be detected, which is according to EU regulations the highest tolerable pesticide concentration in infant food.43 In this respect, the value of the test lies in its potential to also detect phosphorothionates, i.e., the most potent AChE inhibitors. Currently, the only minor drawback of the method is the requirement of expensive NADPH as cofactor. However, research is being carried out to overcome the problem by combining the P450 ΒΜ-3 system with a cofactor recycling system that is immobilized directly on the biosensor.44 ACKNOWLEDGMENT

(39) Nunes, G. S.; Barcelo, D.; Grabaric, B. S.; Diaz-Cruz, J. M.; Ribeiro, M. L. Anal. Chim. Acta 1999, 399, 37-49. (40) Palchetti, I.; Cagnini, A.; Carlo, M. D.; Coppi, C.; Mascini, M.; Turner, A. P. F. Anal. Chim. Acta 1997, 337, 315-21. (41) Pogacnik, L.; Franko, M. Biosens. Bioelectron. 1999, 14, 569-78. (42) Skladal, P.; Nunes, G. S.; Yamanaka, H.; Ribeiro, M. L. Electroanalysis 1997, 9, 1083-7. (43) EC Official J. Eur. Communities 1999, L139, 29-31. (44) Maurer, S. C.; Schulze, H.; Schmid, R. D.; Urlacher, V. Adv. Synth. Catal. 2003, 345, 802-10.

The authors thank the European Union for financial support under Project ACHEB (QLK3-2000-00650).

Received for review October 15, 2003. Accepted January 16, 2004. AC035218T Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

1725