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New tools in diagnosis and biomonitoring of intoxications with organophosphorothioates: Case studies with chlorpyrifos and diazinon
Chemico-Biological Interactions 203 (2013) 96–102
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
New tools in diagnosis and biomonitoring of intoxications with organophosphorothioates: Case studies with chlorpyrifos and diazinon M.J. van der Schans a,⇑, A.G. Hulst a, D. van der Riet – van Oeveren a, D. Noort a, H.P. Benschop a, Ch. Dishovsky b a b
TNO, CBRN Protection, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Military Medical Academy, 3 Georgi Sofiisky Boul., 1606 Sofia, Bulgaria
a r t i c l e
i n f o
Article history: Available online 1 November 2012 Keywords: Pesticides Diagnosis Organophosphates Adducts
a b s t r a c t Organophosphate (OP) pesticides are neurotoxic compounds that are widely used in agriculture. Classical methods for monitoring OP exposure comprise the measurement of intact OP, its metabolites or cholinesterase activity. Newly developed methods focus on the analysis of the OP adduct bound to proteins such as butyrylcholinesterase (BuChE) and albumin. These adducts can be analyzed by means of fluoride reactivation or by analysis with LC–MS/MS of the pepsin or pronase digest of butyrylcholinesterase and albumin, respectively. The utility of these methods is illustrated through the analysis of plasma samples obtained from patients taken 1–49 days after ingestion of the organophosphate pesticides chlorpyrifos and/or diazinon. Thus, in this particular case several independent methodologies were applied to the biomedical samples, all pointing to the same exposure. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The widespread use of organophosphate (OP) pesticides in agriculture and to a lesser extent in households leads each year to millions of intoxications worldwide. Among these pesticides chlorpyrifos alone causes each year at least ten thousand fatalities, mostly due to suicide [1–3]. Medical care of patients severely intoxicated with organophosphate pesticides involves intensive treatment in hospital including lavage, artificial respiration, carbo-chemoperfusion and treatment with specific antidotes, e.g., oximes, atropine and diazepam. In addition to monitoring the general parameters for the health status of the patient, more specific measurements involve analysis of the intact pesticide in blood, measurement of hydrolysis products of the (thio-) phosphates in urine as well as monitoring the degree of inhibition of cholinesterases in blood [4]. Evidently, a rapid and adequate diagnosis that elucidates the identity of the inhibitor and the severity of the intoxication is essential for the choice and dose of the oxime to ensure an optimal treatment of the patient. Moreover in fatal cases of OP intoxications (terrorist attacks, suicide, homicide or poisoning cases), a forensic investigation will be needed, which requires analytical methodology to determine the identity of the toxicant. For the victims of the terrorist attack with the nerve agent sarin in the Tokyo subway we introduced a new approach to diagnose ⇑ Corresponding author. Tel.: +31 888661283. E-mail address: [email protected] (M.J. van der Schans). 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2012.10.014
and monitor the patients, i.e., incubation of plasma samples with fluoride ions, which converts the phosphyl moieties adducted to BuChE into their corresponding phosphofluoridates. The latter product can be analyzed by means of GC-NPD or MS analysis, at levels P10 pg/ml plasma, corresponding with inhibition levels of BuChE P 0.1% [5,6]. It can be envisaged that this approach will also be useful in biomonitoring exposure to OP’s for various environmental public health applications, since bioaccumulation over extended periods of time can be monitored, restricted only by spontaneous reactivation and dealkylation (aging) of the phosphyl moiety and the rate of sequestration of the adducted protein. In addition to fluoride reactivation, we developed a procedure based on LC–MS analysis of the peptide containing the active site serine that results after pepsin digestion of BuChE isolated from plasma. This method enables analysis of both non-aged and aged phosphyl moieties bound to serine [7]. Additionally, we published an analytical method for the determination of organophosphorothioate adducts to albumin [8], while other research groups reported the adducts of organophosphate oxons [9,10]. In the present paper we report on diagnosis and monitoring in two cases of severe phosphorothioate intoxication, i.e., a case of suicidal intoxication with chlorpyrifos and a case of accidental intoxication with a mixture of the latter pesticide and diazinon. We investigated the formation of adducts of the phosphorothioate pesticides and their activated oxon-metabolites with proteins by means of fluoride-induced reactivation and by means of LC–MS analysis of target peptides derived from adducted proteins.
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M.J. van der Schans et al. / Chemico-Biological Interactions 203 (2013) 96–102 Table 1 BuChE activity (U/l) and generated phosphofluoridates after fluoride reactivation in plasma of patients that ingested chloropyrifos (case 1) and a mixture of chlorpyrifos and diazinon (case 2). Case
1 1 2 2
Sample nr.
Period after ingestion (days)
BuChE activity (U/l)
DEFTP (ng/ml)
(nM)
DEFP (ng/ml)
(nM)
1 2 3 4
33 49 1 21
673 2020 170 260
12.1 3.9 327 25.9
70.3 22.7 1901 151
2.75 1.56 26.5 3.80
17.6 10.0 169 24.4
Normal values for BuChE activity are 4200–10,800 U/l.
2. Experimental 2.1. Instrumentation 2.1.1. GC–NPD Fluoridates and intact pesticides were analyzed on a HRGC Mega II 8560 instrument (Fisons Instruments, Milan, Italy) equipped with a nitrogen–phosphorus detector and a split–splitless injector. Oven temperature was 70 °C at the start and elevated with 10 °C/min to 300 °C. Other conditions were as described by van der Schans et al. [11]. 2.1.2. LC–MS/MS LC–MS analyses were performed on a TSQ Quantum Ultra triple quad mass spectrometry instrument from Thermo Scientific (Breda, The Netherlands). The HPLC system was an Acquity system from Waters (Milford, MA, USA). Stationary phase was an Acquity HSS T3 (100 mm 2.1 mm, 1.8 lm particles) from Waters. The mobile phase consisted of a gradient of A: 0.2% formic acid in water and B: 0.2% formic acid in acetonitrile. Gradient program was 0– 100 : 100% A ? 50% A; 10–150 : 50% A ? 100% B. The pump flow was 0.1 ml/min. For the detection of Tyrosine adducts, the MRM transitions m/z 334 ? 260 + 288 and m/z 318 ? 244 + 272 were recorded, corresponding with the P@S and P@O derivatives, respectively. The collision energy was 17 eV. For the FEGSAGAAS nonapeptide, the MRM transitions m/z 932 ? 778 and m/z 904 ? 778 were recorded, corresponding with the P(OEt)2 and P(OEt)(OH) derivatives, respectively. Collision energy was 28 eV. 2.2. Materials Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate, 99.5%], diazinon [O,O-diethyl O-(2-isopropyl-4-
methyl-pyrimidyl) phosphorothioate, 99.5%] were purchased from Chem Service (West Chester, PA, USA). Acetic acid, formic acid, acetonitrile, n-hexane, methanol, potassium fluoride and sodium hydroxide was purchased from Merck, Germany. Pepsin was purchased from Roche Applied Science (Almere, The Netherlands). Pronase type XIV from Streptomyces griseus (E.C. 3.4.24.31) was obtained from Sigma Chemical Company (St Louis, MO, USA). Nexus cartridges were purchased from Varian (Middelburg, The Netherlands). Reference compounds (phosphotyrosine derivatives and phosphylated peptides) were synthesized in-house in mg quantities, following literature procedures. The synthetic compounds were purified to a purity >90% (according to HPLC with UV detection) and their identity was assessed by means of LC–MS. Details on the synthesis of reference peptides can be found in the Supplemental data. 2.3. Methods BuChE activities in plasma samples were measured in the clinical hospital in Sofia on a Hospitex Diagnostics – Screen Master instrument (Florence, Italy) using the BuChE detection kit from Giesse Diagnostics (code 4153, Rome, Italy). Intact pesticides were extracted from plasma (500 ll) with 1 ml n-hexane. The extract was evaporated with a gentle stream of nitrogen and the residue was re-dissolved in 100 ll n-hexane and analyzed with GC-NPD. The fluoride reactivation method was performed as earlier described by Degenhardt et al. [6]. For the analysis of phosphylated BuChE, BuChE was isolated by means of extraction on a procainamide gel followed by digestion with pepsin and analysis with LC–MS [7,12]. For the analysis of phosphylated albumin, albumin was isolated using a HiTrap Blue HP affinity column, followed by digestion with pronase and analysis with LC–MS [8]. Details on the procedures can be found in the Supplemental data. 2.4. Medical treatment of patients Case 1 pertained to a female patient (age 41 years) who attempted suicide by ingestion of an unknown amount of chlorpyrifos. She was treated in the Clinic of Toxicology, MHATEM ‘‘N.I. Pirogov’’, Sofia, Bulgaria. Case 2 involved a male patient (age 66) who ingested accidentally an unknown amount of a mixture of approximately equal amounts of chlorpyrifos and diazinon. He was hospitalized in the Clinic of Toxicology of the Military Medical Academy, also in Sofia.
20 GB (IS)
15 E tO
S P
E tO
O
E tO
F
DEFTP
NPD signal (mV)
P E tO
10
F
DEFP Chlorpyrifos
5
non exposed
0
-5 4
5
6
7
8
Time (min) Fig. 1. GC–NPD Chromatogram of plasma samples that were processed according to the fluoride reactivation method. Lower trace: non-exposed plasma sample, upper trace: plasma sample from a patient that had ingested chlorpyrifos (case 1, sample was taken 33 days after ingestion). Trace line of exposed sample was moved upwards with 5 mV in order to improve visibility in the overlay of the two chromatograms.
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A
95
NL: 5.00E3 m/z= 777.80-778.80 F: + c ESI sid=10.00 SRM ms2 904.400 [602.095-602.105, 673.095-673.105, 778.295-778.305] MS 09ACM073_6
90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 11.83
10
13.63
5 0.34
0 0
2.21 2.28
11.46 8.83 9.95 11.13
3.37 4.26 5.37 5.91 6.49 7.11
2
4
6
8
10
14.45
16.28
16.24 14.72
13.39 12.57
12
14
17.04 17.89
16
18
19.34
20.67
20
Time (min) RT: 0.00 - 21.00 13.47
100
NL: 5.07E3 m/z= 777.80-778.80 F: + c ESI sid=10.00 SRM ms2 904.400 [602.095-602.105, 673.095-673.105, 778.295-778.305] MS 09ACM073_1
B
95 90
13.52
85 80
FGESAGAAS
75
O
70
HO P O
65
OC 2H5
60 55 50 45 40 35 30 25
11.76
20 15
16.07
10 13.75
5 0.81 1.27 1.98
0 0
2
3.49
5.76
4
5.81
6
7.72
7.84
11.91 8.28
8
15.09
10.31 11.21
10 Time (min)
12
14
17.00
16
18.43
18
19.52 20.88
20
Fig. 2. LC–MS–MS ion chromatogram in MRM mode (m/z 904 ? 778) of nonapeptide FGES⁄AGAAS, with S⁄ the active site serine adducted with a mono-ethylphosphate moiety, in a pepsin digest of BuChE isolated from plasma. A: Non-exposed plasma, B: plasma from patient who had ingested chlorpyrifos (see Fig. 1).
Both patients were lavaged immediately after hospitalization and were submitted to carbo-perfusion for 4–6 h on the first day, whereas the male patient (case 2) was also carbo-perfused
on the second day. In addition to atropine and diazepam, they received daily for six consecutive days three 250 mg iv-dosages of Toxogonin (obidoxim). With their written consent, blood
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M.J. van der Schans et al. / Chemico-Biological Interactions 203 (2013) 96–102 RT: 0.00 - 15.00 NL: 5.20E4 m/z= 259.40-260.40+ 287.50-288.50 F: + c ESI SRM ms2 334.100 [259.850-259.950, 287.950-288.050] MS 0836MS005
100 95
A
90 85 80 75
Relative Abundance
70 65 60 55 50 45 40 35 30 25 20
4.69
15
4.74
4.76
10 5
2.77 2.05 2.19
0.84
0
0
1
2
5.48
4.04 4.18 3.79
5.63 6.14
4
3
7.03
6
5
7.14
7.97 8.60 9.23
8 7 Time (min)
10.34
10
9
11.66
11.99 12.47
11
14.00 14.15
13
12
14
15
RT: 0.00 - 15.00 NL: 5.20E4 m/z= 259.40-260.40+ 287.50-288.50 F: + c ESI SRM ms2 334.100 [259.850-259.950, 287.950-288.050] MS 0836MS006
9.05
100 95
B
90 85 80
H
75
H2 N C COOH
70
CH 2
Relative Abundance
65 60 55
H H O H 3 C C O P O C CH3
50 45
H
H
S
40 35 30 25 9.19
20 4.62
15
9.22
4.66
4.70
10
4.22
5 0.31
0 0
3.70 2.17 2.87 2.96 1.04 1.60
1
2
3
4.18
5.37
4
5
5.40 5.49
6
6.03 6.94 6.99 7.33
7
9.31 9.53 9.98
8.39
8
9
10
11.17 11.72
11
12
12.07
13
13.88 14.20
14
15
Time (min)
Fig. 3. LC–MS–MS ion chromatogram in MRM mode (m/z 334 ? 260 + 288 and m/z 318 ? 244 + 272) of the tyrosine diethylphosphothioate and tyrosine diethylphosphoro adduct in a pronase digest of albumin isolated from a plasma samples of a patient that had ingested chlorpyrifos (see Fig. 1). A: m/z 334 ? 260 + 288, non-exposed plasma; B: m/z 334 ? 260 + 288, plasma from patient C: m/z 318 ? 244 + 27,2 non-exposed sample and D: m/z 318 ? 244 + 272, plasma from patient.
samples for investigative purposes were taken by venous puncture and were collected in heparinised tubes. These samples were taken at 33 and 49 days and 1 day and 21 days for case 1 and case 2 respectively. These incidents occurred in the year 2006. Small aliquots of the plasma samples were frozen and shipped to TNO in The Netherlands in 2008 for further analysis.
3. Results 3.1. BuChE activity Table 1 shows the BuChE activity status of the two cases that presented in this study. Both patients were released from hospital
100
M.J. van der Schans et al. / Chemico-Biological Interactions 203 (2013) 96–102 RT: 0.00 - 15.00 NL: 4.40E5 m/z= 243.50-244.50+ 271.50-272.50 F: + c ESI SRM ms2 318.100 [243.950-244.050, 271.950-272.050] MS 0836MS005
100 95
C
90 85 80 75 70
Relative Abundance
65 60 55 50 45 40 35 30 25 20 15 10 5 0
1
2
5.52 6.48 6.54 6.59 7.16
3.73 4.68 4.70
0.81 2.03 2.21 2.53
0
3
4
5
6
8.64
7 8 Time (min)
9.54 9.90 10.60 11.39 11.91 12.91 13.26 13.68 14.49
9
10
11
12
13
14
15
RT: 0.00 - 15.00 NL: 4.48E5 m/z= 243.50-244.50+ 271.50-272.50 F: + c ESI SRM ms2 318.100 [243.950-244.050, 271.950-272.050] MS 0836MS006
6.44
100 95
D
90
H
85
H2N C COOH
80
CH2
75 70
Relative Abundance
65
O H H H3C C O P O C CH3 O H H
60 55 50 45 40 35 30 25 20 15 10
6.66
5 0.60 0.98 2.04
0 0
1
2
3
6.74
4.57
3.70
2.27 2.69
4
5.39 5.53
5
6
7
7.05
7.65
9.20
8
9
Time (min)
Fig. 3. (continued)
10.11 10.67 11.35
10
11
12.16
12
13.29 13.78 14.41
13
14
15
101
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in a good clinical state of health, although the BuChE activity was still abnormally low for the male patient (case 2). 3.2. Fluoride-induced reactivation of plasma samples The four plasma samples that were shipped to TNO were processed according to the fluoride reactivation technique and analyzed with GC–NPD. Two peaks were observed in the gas chromatogram, which could be identified as O,O-diethyl phosphorofluoridate (DEFP) and O,O-diethyl phosphorofluoridothioate (DEFTP). Fig. 1 shows a representative chromatogram whereas quantitative data are collected in Table 1. Presumably, the source of the fluoridates can be twofold: (i) regeneration of an adduct, or (ii) direct conversion of the pesticide or its oxon itself. Only for sample #3 it was verified that it contained chlorpyrifos and diazinon at levels of 6.2 and 2.4 lg/ml respectively, which corresponds with 17.6 and 7.8 lM. The other samples did not contain pesticide, which means that the fluoridates cannot be formed from the pesticides itself. In order to elucidate the origin of the fluoridates and obtain a better insight into the identity of the adducts the available samples were processed and analyzed with LC–MS, as described below. 3.3. Mass spectrometric analysis of phosphylated butyrylcholinesterase The four plasma samples were processed according to the described methodology for mass spectrometric analysis of the BuChE-adducts. BuChE was isolated by means of extraction with
In vivo reaction
“Fluoride reactivation”
procainamide gel and digested with pepsin for LC–MS analysis in order to analyze the nonapeptide FGES⁄AGAAS with S⁄ representing the serine-198 containing adducted serine, pertaining to the active site of the enzyme [7,12]. It appeared that in all samples only the mono-dealkylated phosphoryl moiety bound to serine could be observed. Fig. 2 shows a representative ion chromatogram. Presumably, the thioate pesticide had been converted into the oxon analog, which inhibited BuChE. Subsequently, aging had taken place, which was almost complete within 1 day after poisoning had occurred [13]. Moreover, these patients received a treatment with obidoxime, which means that the non-aged adduct was removed from BuChE and the remaining levels of the non-aged adduct were below the detection limit of the analysis. Therefore only the aged from, which cannot be reactivated, was detected in this assay. This result indicates that inhibited BuChE was not the source of DEFP that was detected in the samples that were processed according to the fluoride reactivation technique. 3.4. Mass spectrometric analysis of phosphylated albumin Albumin from plasma samples of case 1 taken at 33 and 49 days after poisoning was isolated by means of a HiTrap Blue HP affinity column and digested with pronase according to our recently published procedure [8] for LC–MS/MS analysis of Tyr-411 adducted at its phenolic function with a O,O-diethyl phoshorothionyl moiety. In both samples the fore mentioned adduct could be detected and Fig. 3 shows a representative ion-chromatogram. The MRM transition m/z 318 ? 244 + 272 was also acquired for analysis of the O,O-diethylphosphoro adduct to albumin. This adduct was also
Pronasedigestion
Pepsindigestion
S H5C2O
P OC2H5 OH
S H5C2O
P OC2H5 F
DETP S H5C2O P OC2H5 O Cl N
Albumin
LVRYTKKVPQ O H5C2O P S
H5C2O
OC2H5
OC2H5
Cl
H5C2O
Cl
DEFTP
Y O P S
Tyr-OP=S
Y O P O OC2H5
Tyr-OP=O
Chlorpyrifos
P450
LVRYTKKVPQ O H5C2O P O
O H5C2O P OC H 2 5 F
OC2H5
DEFP
O H5C2O P OC2H5 O Cl
FGESAGAAS
BuChE
N Cl Cl
O H5C2O
H5C2O
O P O OC2H5
ageing
P OC2H5 OH
DEP
Chlorpyrifos-oxon
FGESAGAAS O H5C2O P O OC2H5
FGESAGAAS O HO P O OC2H5
FGESAGAAS O HO P
O
OC 2H 5
Fig. 4. Elimination pathways of chlorpyrifos and analytical methods leading to biomarkers of exposure. Note that formed DEFP can have four different sources. Dashed lines indicate potential pathways. Solid lines indicate pathways that were confirmed by the current study through analysis of the plasma sample of the victim that was exposed to chlorpyrifos (see Fig. 1). The black figures and arrows represent the in vivo metabolic pathways, the red arrows show the biomarkers that can be detected using pepsin digestion of BuChE; purple arrows indicate the pronase assay of albumin and green arrows represent the formation of DEFTP and DEFP in the fluoride reactivation assay. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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detected and indicates that this adduct can be the source for the formation of DEFP in the fluoride reactivation assay. This assay was not performed for samples from case 2 since insufficient sample was available. 3.5. Additional explanation for the formation of DEFP in the fluoride reactivation assay The fluoride reactivation assay revealed the formation of the DEFP and DEFTP fluoridates in all plasma samples. It seems that DEFP and DEFTP were both released from albumin in the fluoride reactivation assay. However, during some exploratory experiments, it was found that DEFP can also be formed from the DEFTP thio analog through a thion–thiolo rearrangement. In order to verify this hypothesis, some control experiments were performed. Firstly chlorpyrifos (1000 lM) was incubated in plasma to induce the formation of adducts. After 24 h of incubation the excess of chlorpyrifos was extracted with hexane until chlorpyrifos was not detected in the extract. (Extracts were analyzed with GC– NPD). After these extractions, the sample was processed and analyzed according to the fluoride reactivation technique. Both DEFTP and DEFP could be detected at concentrations of 0.13 and 0.1 lM respectively. In a second experiment, a solution of DEFTP-Tyr adduct at a concentration of 0.15 lM was processed and analyzed following the fluoride reactivation technique. Both fluoride reactivation products DEFTP and DEFP could be detected with GC–NPD at concentrations of 0.018 and 0.014 lM respectively. From these two experiments it can be concluded that upon incubation with fluoride ions, the DEFP product can also formed from the O,Odiethyl phoshorothionyl adduct. Fig. 4 summarizes the in vivo pathways of chlorpyrifos and the following conversion of the adduct into the biomarker of exposure, as result of the sample processing for the diverse analytical methods. Fig. 4 illustrates that DEFP can be formed in the fluoride reactivation assay from four different sources. 4. Conclusion Verification of OP exposure can have several purposes, such as: (i) monitoring health status in order to optimize therapy and (ii) forensic purposes in case of suspected malicious usage of the agent. In addition to the classical methods such as cholinesterase activity, intact agent and metabolites measurements, determination of adducts can have added value. The fluoride reactivation method results in a collection of all fluoride derivatives of the organophosphates from sources varying from the original pesticide to adducts with BuChE, albumin and as yet unknown binding sites, which yields valuable insight into the total body burden of organophosphate anticholinesterases in individuals. LC–MS/MS analysis of adducts to proteins provides a more detailed insight in the origin of these adducts, it is also able to detect the aged OP-adduct which would not be detected with the fluoride reactivation method. Moreover, it helps the interpretation of the results that have been obtained with the fluoride reactivation technique. In this case the formation of DEFP might suggest that the adduct originated from BuChE and would suggest therapy with oximes, to reactivate acetylcholinesterase, might still be effective. However, the analysis of phosphylated BuChE brought up another perspective and revealed that the reactivatable adduct
DEFP was not present on BuChE. In conclusion, the current paper shows that multiple analytical techniques are available for the verification of exposure to an OP pesticide, which enable the retrospective verification of that exposure. In this case the retrospectivity was that long, that even at 49 days after the ingestion of the pesticides, the adducts could still be detected. Therefore these methods can be very useful for research in the field of molecular epidemiology. Finally, the multiple, orthogonal methods pointing unequivocally to a particular exposure, is also highly relevant from a forensic point of view.
Conflict of interest The authors declare that there is no conflict of interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2012.10.014. References [1] F. Eyer, D.M. Roberts, N.A. Buckley, M. Eddleston, H. Thiermann, F. Worek, P. Eyer, Extreme variability in the formation of chlorpyrifos oxon (CPO) in patients poisoned by chlorpyrifos (CPS), Biochem. Pharmacol. 78 (2009) 531– 537. [2] R. Heilmair, F. Eyer, P. Eyer, Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma, Toxicol. Lett. 181 (2008) 19–24. [3] M. Eddleston, P. Eyer, F. Worek, F. Mohamed, L. Senarathna, L. von Meyer, Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study, Lancet 366 (2005) 1452–1459. [4] D.B. Barr, J. Angerer, Potential uses of biomonitoring data: a case study using the organophosphorus pesticides chlorpyrifos and malathion, Environ. Health Perspect. 114 (2006) 1763–1769. [5] M. Polhuijs, J.P. Langenberg, H.P. Benschop, New method for retrospective detection of exposure to organophosphorus anticholinesterases: application to alleged sarin victims of Japanese terrorists, Toxicol. Appl. Pharmacol. 146 (1997) 156–161. [6] C.E.A.M. Degenhardt, K. Pleijsier, M.J. van der Schans, J.P. Langenberg, K.E. Preston, M.I. Solano, V.L. Maggio, J.R. Barr, Improvements of the fluoride reactivation method for the verification of nerve agent exposure, J. Anal. Toxicol. 28 (2004) 364–371. [7] A. Fidder, A.G. Hulst, D. Noort, R. de Ruiter, M.J. van der Schans, H.P. Benschop, J.P. Langenberg, Retrospective detection of exposure to organophosphorus anti-cholinesterases: mass spectrometric analysis of phosphylated human butyrylcholinesterase, Chem. Res. Toxicol. 15 (2002) 582–590. [8] D. Noort, A.G. Hulst, A. van Zuylen, E. van Rijssel, M.J. van der Schans, Covalent binding of organophosphorothioates to albumin: a new perspective for OPpesticide biomonitoring?, Arch Toxicol. 83 (2009) 1031–1036. [9] B. Li, I. Ricordel, L.M. Schopfer, F. Baud, B. Megarbane, F. Nachon, P. Masson, O. Lockridge, Detection of adduct on tyrosine-411 of albumin in humans poisoned by dichlorvos, Toxicol. Sci. 116 (2010) 23–31. [10] B. Li, I. Ricordel, L.M. Schopfer, F. Baud, B. Mégarbane, P. Masson, O. Lockridge, Dichlorvos, chlorpyrifos-oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose, J. Appl. Toxicol. 30 (2010) 559–565. [11] M.J. van der Schans, B.J. Lander, H. van der Wiel, J.P. Langenberg, H.P. Benschop, Toxicokinetics of the nerve agent (+/)-VX in anesthetized and atropinized hairless guinea pigs and marmosets after intravenous and percutaneous administration, Toxicol. Appl. Pharmacol. 191 (2003) 48–62. [12] M.J. van der Schans, A. Fidder, D. van Oeveren, A.G. Hulst, D. Noort, Verification of exposure to cholinesterase inhibitors: generic detection of OPCW Schedule 1 nerve agent adducts to human butyrylcholinesterase, J. Anal. Toxicol. 31 (2008) 125–130. [13] N. Aurbek, H. Thiermann, F. Eyer, P. Eyer, Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: a kinetic analysis, Toxicology 229 (2009) 133– 139.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
New tools in diagnosis and biomonitoring of intoxications with organophosphorothioates: Case studies with chlorpyrifos and diazinon M.J. van der Schans a,⇑, A.G. Hulst a, D. van der Riet – van Oeveren a, D. Noort a, H.P. Benschop a, Ch. Dishovsky b a b
TNO, CBRN Protection, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Military Medical Academy, 3 Georgi Sofiisky Boul., 1606 Sofia, Bulgaria
a r t i c l e
i n f o
Article history: Available online 1 November 2012 Keywords: Pesticides Diagnosis Organophosphates Adducts
a b s t r a c t Organophosphate (OP) pesticides are neurotoxic compounds that are widely used in agriculture. Classical methods for monitoring OP exposure comprise the measurement of intact OP, its metabolites or cholinesterase activity. Newly developed methods focus on the analysis of the OP adduct bound to proteins such as butyrylcholinesterase (BuChE) and albumin. These adducts can be analyzed by means of fluoride reactivation or by analysis with LC–MS/MS of the pepsin or pronase digest of butyrylcholinesterase and albumin, respectively. The utility of these methods is illustrated through the analysis of plasma samples obtained from patients taken 1–49 days after ingestion of the organophosphate pesticides chlorpyrifos and/or diazinon. Thus, in this particular case several independent methodologies were applied to the biomedical samples, all pointing to the same exposure. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The widespread use of organophosphate (OP) pesticides in agriculture and to a lesser extent in households leads each year to millions of intoxications worldwide. Among these pesticides chlorpyrifos alone causes each year at least ten thousand fatalities, mostly due to suicide [1–3]. Medical care of patients severely intoxicated with organophosphate pesticides involves intensive treatment in hospital including lavage, artificial respiration, carbo-chemoperfusion and treatment with specific antidotes, e.g., oximes, atropine and diazepam. In addition to monitoring the general parameters for the health status of the patient, more specific measurements involve analysis of the intact pesticide in blood, measurement of hydrolysis products of the (thio-) phosphates in urine as well as monitoring the degree of inhibition of cholinesterases in blood [4]. Evidently, a rapid and adequate diagnosis that elucidates the identity of the inhibitor and the severity of the intoxication is essential for the choice and dose of the oxime to ensure an optimal treatment of the patient. Moreover in fatal cases of OP intoxications (terrorist attacks, suicide, homicide or poisoning cases), a forensic investigation will be needed, which requires analytical methodology to determine the identity of the toxicant. For the victims of the terrorist attack with the nerve agent sarin in the Tokyo subway we introduced a new approach to diagnose ⇑ Corresponding author. Tel.: +31 888661283. E-mail address: [email protected] (M.J. van der Schans). 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2012.10.014
and monitor the patients, i.e., incubation of plasma samples with fluoride ions, which converts the phosphyl moieties adducted to BuChE into their corresponding phosphofluoridates. The latter product can be analyzed by means of GC-NPD or MS analysis, at levels P10 pg/ml plasma, corresponding with inhibition levels of BuChE P 0.1% [5,6]. It can be envisaged that this approach will also be useful in biomonitoring exposure to OP’s for various environmental public health applications, since bioaccumulation over extended periods of time can be monitored, restricted only by spontaneous reactivation and dealkylation (aging) of the phosphyl moiety and the rate of sequestration of the adducted protein. In addition to fluoride reactivation, we developed a procedure based on LC–MS analysis of the peptide containing the active site serine that results after pepsin digestion of BuChE isolated from plasma. This method enables analysis of both non-aged and aged phosphyl moieties bound to serine [7]. Additionally, we published an analytical method for the determination of organophosphorothioate adducts to albumin [8], while other research groups reported the adducts of organophosphate oxons [9,10]. In the present paper we report on diagnosis and monitoring in two cases of severe phosphorothioate intoxication, i.e., a case of suicidal intoxication with chlorpyrifos and a case of accidental intoxication with a mixture of the latter pesticide and diazinon. We investigated the formation of adducts of the phosphorothioate pesticides and their activated oxon-metabolites with proteins by means of fluoride-induced reactivation and by means of LC–MS analysis of target peptides derived from adducted proteins.
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M.J. van der Schans et al. / Chemico-Biological Interactions 203 (2013) 96–102 Table 1 BuChE activity (U/l) and generated phosphofluoridates after fluoride reactivation in plasma of patients that ingested chloropyrifos (case 1) and a mixture of chlorpyrifos and diazinon (case 2). Case
1 1 2 2
Sample nr.
Period after ingestion (days)
BuChE activity (U/l)
DEFTP (ng/ml)
(nM)
DEFP (ng/ml)
(nM)
1 2 3 4
33 49 1 21
673 2020 170 260
12.1 3.9 327 25.9
70.3 22.7 1901 151
2.75 1.56 26.5 3.80
17.6 10.0 169 24.4
Normal values for BuChE activity are 4200–10,800 U/l.
2. Experimental 2.1. Instrumentation 2.1.1. GC–NPD Fluoridates and intact pesticides were analyzed on a HRGC Mega II 8560 instrument (Fisons Instruments, Milan, Italy) equipped with a nitrogen–phosphorus detector and a split–splitless injector. Oven temperature was 70 °C at the start and elevated with 10 °C/min to 300 °C. Other conditions were as described by van der Schans et al. [11]. 2.1.2. LC–MS/MS LC–MS analyses were performed on a TSQ Quantum Ultra triple quad mass spectrometry instrument from Thermo Scientific (Breda, The Netherlands). The HPLC system was an Acquity system from Waters (Milford, MA, USA). Stationary phase was an Acquity HSS T3 (100 mm 2.1 mm, 1.8 lm particles) from Waters. The mobile phase consisted of a gradient of A: 0.2% formic acid in water and B: 0.2% formic acid in acetonitrile. Gradient program was 0– 100 : 100% A ? 50% A; 10–150 : 50% A ? 100% B. The pump flow was 0.1 ml/min. For the detection of Tyrosine adducts, the MRM transitions m/z 334 ? 260 + 288 and m/z 318 ? 244 + 272 were recorded, corresponding with the P@S and P@O derivatives, respectively. The collision energy was 17 eV. For the FEGSAGAAS nonapeptide, the MRM transitions m/z 932 ? 778 and m/z 904 ? 778 were recorded, corresponding with the P(OEt)2 and P(OEt)(OH) derivatives, respectively. Collision energy was 28 eV. 2.2. Materials Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate, 99.5%], diazinon [O,O-diethyl O-(2-isopropyl-4-
methyl-pyrimidyl) phosphorothioate, 99.5%] were purchased from Chem Service (West Chester, PA, USA). Acetic acid, formic acid, acetonitrile, n-hexane, methanol, potassium fluoride and sodium hydroxide was purchased from Merck, Germany. Pepsin was purchased from Roche Applied Science (Almere, The Netherlands). Pronase type XIV from Streptomyces griseus (E.C. 3.4.24.31) was obtained from Sigma Chemical Company (St Louis, MO, USA). Nexus cartridges were purchased from Varian (Middelburg, The Netherlands). Reference compounds (phosphotyrosine derivatives and phosphylated peptides) were synthesized in-house in mg quantities, following literature procedures. The synthetic compounds were purified to a purity >90% (according to HPLC with UV detection) and their identity was assessed by means of LC–MS. Details on the synthesis of reference peptides can be found in the Supplemental data. 2.3. Methods BuChE activities in plasma samples were measured in the clinical hospital in Sofia on a Hospitex Diagnostics – Screen Master instrument (Florence, Italy) using the BuChE detection kit from Giesse Diagnostics (code 4153, Rome, Italy). Intact pesticides were extracted from plasma (500 ll) with 1 ml n-hexane. The extract was evaporated with a gentle stream of nitrogen and the residue was re-dissolved in 100 ll n-hexane and analyzed with GC-NPD. The fluoride reactivation method was performed as earlier described by Degenhardt et al. [6]. For the analysis of phosphylated BuChE, BuChE was isolated by means of extraction on a procainamide gel followed by digestion with pepsin and analysis with LC–MS [7,12]. For the analysis of phosphylated albumin, albumin was isolated using a HiTrap Blue HP affinity column, followed by digestion with pronase and analysis with LC–MS [8]. Details on the procedures can be found in the Supplemental data. 2.4. Medical treatment of patients Case 1 pertained to a female patient (age 41 years) who attempted suicide by ingestion of an unknown amount of chlorpyrifos. She was treated in the Clinic of Toxicology, MHATEM ‘‘N.I. Pirogov’’, Sofia, Bulgaria. Case 2 involved a male patient (age 66) who ingested accidentally an unknown amount of a mixture of approximately equal amounts of chlorpyrifos and diazinon. He was hospitalized in the Clinic of Toxicology of the Military Medical Academy, also in Sofia.
20 GB (IS)
15 E tO
S P
E tO
O
E tO
F
DEFTP
NPD signal (mV)
P E tO
10
F
DEFP Chlorpyrifos
5
non exposed
0
-5 4
5
6
7
8
Time (min) Fig. 1. GC–NPD Chromatogram of plasma samples that were processed according to the fluoride reactivation method. Lower trace: non-exposed plasma sample, upper trace: plasma sample from a patient that had ingested chlorpyrifos (case 1, sample was taken 33 days after ingestion). Trace line of exposed sample was moved upwards with 5 mV in order to improve visibility in the overlay of the two chromatograms.
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A
95
NL: 5.00E3 m/z= 777.80-778.80 F: + c ESI sid=10.00 SRM ms2 904.400 [602.095-602.105, 673.095-673.105, 778.295-778.305] MS 09ACM073_6
90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 11.83
10
13.63
5 0.34
0 0
2.21 2.28
11.46 8.83 9.95 11.13
3.37 4.26 5.37 5.91 6.49 7.11
2
4
6
8
10
14.45
16.28
16.24 14.72
13.39 12.57
12
14
17.04 17.89
16
18
19.34
20.67
20
Time (min) RT: 0.00 - 21.00 13.47
100
NL: 5.07E3 m/z= 777.80-778.80 F: + c ESI sid=10.00 SRM ms2 904.400 [602.095-602.105, 673.095-673.105, 778.295-778.305] MS 09ACM073_1
B
95 90
13.52
85 80
FGESAGAAS
75
O
70
HO P O
65
OC 2H5
60 55 50 45 40 35 30 25
11.76
20 15
16.07
10 13.75
5 0.81 1.27 1.98
0 0
2
3.49
5.76
4
5.81
6
7.72
7.84
11.91 8.28
8
15.09
10.31 11.21
10 Time (min)
12
14
17.00
16
18.43
18
19.52 20.88
20
Fig. 2. LC–MS–MS ion chromatogram in MRM mode (m/z 904 ? 778) of nonapeptide FGES⁄AGAAS, with S⁄ the active site serine adducted with a mono-ethylphosphate moiety, in a pepsin digest of BuChE isolated from plasma. A: Non-exposed plasma, B: plasma from patient who had ingested chlorpyrifos (see Fig. 1).
Both patients were lavaged immediately after hospitalization and were submitted to carbo-perfusion for 4–6 h on the first day, whereas the male patient (case 2) was also carbo-perfused
on the second day. In addition to atropine and diazepam, they received daily for six consecutive days three 250 mg iv-dosages of Toxogonin (obidoxim). With their written consent, blood
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100 95
A
90 85 80 75
Relative Abundance
70 65 60 55 50 45 40 35 30 25 20
4.69
15
4.74
4.76
10 5
2.77 2.05 2.19
0.84
0
0
1
2
5.48
4.04 4.18 3.79
5.63 6.14
4
3
7.03
6
5
7.14
7.97 8.60 9.23
8 7 Time (min)
10.34
10
9
11.66
11.99 12.47
11
14.00 14.15
13
12
14
15
RT: 0.00 - 15.00 NL: 5.20E4 m/z= 259.40-260.40+ 287.50-288.50 F: + c ESI SRM ms2 334.100 [259.850-259.950, 287.950-288.050] MS 0836MS006
9.05
100 95
B
90 85 80
H
75
H2 N C COOH
70
CH 2
Relative Abundance
65 60 55
H H O H 3 C C O P O C CH3
50 45
H
H
S
40 35 30 25 9.19
20 4.62
15
9.22
4.66
4.70
10
4.22
5 0.31
0 0
3.70 2.17 2.87 2.96 1.04 1.60
1
2
3
4.18
5.37
4
5
5.40 5.49
6
6.03 6.94 6.99 7.33
7
9.31 9.53 9.98
8.39
8
9
10
11.17 11.72
11
12
12.07
13
13.88 14.20
14
15
Time (min)
Fig. 3. LC–MS–MS ion chromatogram in MRM mode (m/z 334 ? 260 + 288 and m/z 318 ? 244 + 272) of the tyrosine diethylphosphothioate and tyrosine diethylphosphoro adduct in a pronase digest of albumin isolated from a plasma samples of a patient that had ingested chlorpyrifos (see Fig. 1). A: m/z 334 ? 260 + 288, non-exposed plasma; B: m/z 334 ? 260 + 288, plasma from patient C: m/z 318 ? 244 + 27,2 non-exposed sample and D: m/z 318 ? 244 + 272, plasma from patient.
samples for investigative purposes were taken by venous puncture and were collected in heparinised tubes. These samples were taken at 33 and 49 days and 1 day and 21 days for case 1 and case 2 respectively. These incidents occurred in the year 2006. Small aliquots of the plasma samples were frozen and shipped to TNO in The Netherlands in 2008 for further analysis.
3. Results 3.1. BuChE activity Table 1 shows the BuChE activity status of the two cases that presented in this study. Both patients were released from hospital
100
M.J. van der Schans et al. / Chemico-Biological Interactions 203 (2013) 96–102 RT: 0.00 - 15.00 NL: 4.40E5 m/z= 243.50-244.50+ 271.50-272.50 F: + c ESI SRM ms2 318.100 [243.950-244.050, 271.950-272.050] MS 0836MS005
100 95
C
90 85 80 75 70
Relative Abundance
65 60 55 50 45 40 35 30 25 20 15 10 5 0
1
2
5.52 6.48 6.54 6.59 7.16
3.73 4.68 4.70
0.81 2.03 2.21 2.53
0
3
4
5
6
8.64
7 8 Time (min)
9.54 9.90 10.60 11.39 11.91 12.91 13.26 13.68 14.49
9
10
11
12
13
14
15
RT: 0.00 - 15.00 NL: 4.48E5 m/z= 243.50-244.50+ 271.50-272.50 F: + c ESI SRM ms2 318.100 [243.950-244.050, 271.950-272.050] MS 0836MS006
6.44
100 95
D
90
H
85
H2N C COOH
80
CH2
75 70
Relative Abundance
65
O H H H3C C O P O C CH3 O H H
60 55 50 45 40 35 30 25 20 15 10
6.66
5 0.60 0.98 2.04
0 0
1
2
3
6.74
4.57
3.70
2.27 2.69
4
5.39 5.53
5
6
7
7.05
7.65
9.20
8
9
Time (min)
Fig. 3. (continued)
10.11 10.67 11.35
10
11
12.16
12
13.29 13.78 14.41
13
14
15
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in a good clinical state of health, although the BuChE activity was still abnormally low for the male patient (case 2). 3.2. Fluoride-induced reactivation of plasma samples The four plasma samples that were shipped to TNO were processed according to the fluoride reactivation technique and analyzed with GC–NPD. Two peaks were observed in the gas chromatogram, which could be identified as O,O-diethyl phosphorofluoridate (DEFP) and O,O-diethyl phosphorofluoridothioate (DEFTP). Fig. 1 shows a representative chromatogram whereas quantitative data are collected in Table 1. Presumably, the source of the fluoridates can be twofold: (i) regeneration of an adduct, or (ii) direct conversion of the pesticide or its oxon itself. Only for sample #3 it was verified that it contained chlorpyrifos and diazinon at levels of 6.2 and 2.4 lg/ml respectively, which corresponds with 17.6 and 7.8 lM. The other samples did not contain pesticide, which means that the fluoridates cannot be formed from the pesticides itself. In order to elucidate the origin of the fluoridates and obtain a better insight into the identity of the adducts the available samples were processed and analyzed with LC–MS, as described below. 3.3. Mass spectrometric analysis of phosphylated butyrylcholinesterase The four plasma samples were processed according to the described methodology for mass spectrometric analysis of the BuChE-adducts. BuChE was isolated by means of extraction with
In vivo reaction
“Fluoride reactivation”
procainamide gel and digested with pepsin for LC–MS analysis in order to analyze the nonapeptide FGES⁄AGAAS with S⁄ representing the serine-198 containing adducted serine, pertaining to the active site of the enzyme [7,12]. It appeared that in all samples only the mono-dealkylated phosphoryl moiety bound to serine could be observed. Fig. 2 shows a representative ion chromatogram. Presumably, the thioate pesticide had been converted into the oxon analog, which inhibited BuChE. Subsequently, aging had taken place, which was almost complete within 1 day after poisoning had occurred [13]. Moreover, these patients received a treatment with obidoxime, which means that the non-aged adduct was removed from BuChE and the remaining levels of the non-aged adduct were below the detection limit of the analysis. Therefore only the aged from, which cannot be reactivated, was detected in this assay. This result indicates that inhibited BuChE was not the source of DEFP that was detected in the samples that were processed according to the fluoride reactivation technique. 3.4. Mass spectrometric analysis of phosphylated albumin Albumin from plasma samples of case 1 taken at 33 and 49 days after poisoning was isolated by means of a HiTrap Blue HP affinity column and digested with pronase according to our recently published procedure [8] for LC–MS/MS analysis of Tyr-411 adducted at its phenolic function with a O,O-diethyl phoshorothionyl moiety. In both samples the fore mentioned adduct could be detected and Fig. 3 shows a representative ion-chromatogram. The MRM transition m/z 318 ? 244 + 272 was also acquired for analysis of the O,O-diethylphosphoro adduct to albumin. This adduct was also
Pronasedigestion
Pepsindigestion
S H5C2O
P OC2H5 OH
S H5C2O
P OC2H5 F
DETP S H5C2O P OC2H5 O Cl N
Albumin
LVRYTKKVPQ O H5C2O P S
H5C2O
OC2H5
OC2H5
Cl
H5C2O
Cl
DEFTP
Y O P S
Tyr-OP=S
Y O P O OC2H5
Tyr-OP=O
Chlorpyrifos
P450
LVRYTKKVPQ O H5C2O P O
O H5C2O P OC H 2 5 F
OC2H5
DEFP
O H5C2O P OC2H5 O Cl
FGESAGAAS
BuChE
N Cl Cl
O H5C2O
H5C2O
O P O OC2H5
ageing
P OC2H5 OH
DEP
Chlorpyrifos-oxon
FGESAGAAS O H5C2O P O OC2H5
FGESAGAAS O HO P O OC2H5
FGESAGAAS O HO P
O
OC 2H 5
Fig. 4. Elimination pathways of chlorpyrifos and analytical methods leading to biomarkers of exposure. Note that formed DEFP can have four different sources. Dashed lines indicate potential pathways. Solid lines indicate pathways that were confirmed by the current study through analysis of the plasma sample of the victim that was exposed to chlorpyrifos (see Fig. 1). The black figures and arrows represent the in vivo metabolic pathways, the red arrows show the biomarkers that can be detected using pepsin digestion of BuChE; purple arrows indicate the pronase assay of albumin and green arrows represent the formation of DEFTP and DEFP in the fluoride reactivation assay. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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detected and indicates that this adduct can be the source for the formation of DEFP in the fluoride reactivation assay. This assay was not performed for samples from case 2 since insufficient sample was available. 3.5. Additional explanation for the formation of DEFP in the fluoride reactivation assay The fluoride reactivation assay revealed the formation of the DEFP and DEFTP fluoridates in all plasma samples. It seems that DEFP and DEFTP were both released from albumin in the fluoride reactivation assay. However, during some exploratory experiments, it was found that DEFP can also be formed from the DEFTP thio analog through a thion–thiolo rearrangement. In order to verify this hypothesis, some control experiments were performed. Firstly chlorpyrifos (1000 lM) was incubated in plasma to induce the formation of adducts. After 24 h of incubation the excess of chlorpyrifos was extracted with hexane until chlorpyrifos was not detected in the extract. (Extracts were analyzed with GC– NPD). After these extractions, the sample was processed and analyzed according to the fluoride reactivation technique. Both DEFTP and DEFP could be detected at concentrations of 0.13 and 0.1 lM respectively. In a second experiment, a solution of DEFTP-Tyr adduct at a concentration of 0.15 lM was processed and analyzed following the fluoride reactivation technique. Both fluoride reactivation products DEFTP and DEFP could be detected with GC–NPD at concentrations of 0.018 and 0.014 lM respectively. From these two experiments it can be concluded that upon incubation with fluoride ions, the DEFP product can also formed from the O,Odiethyl phoshorothionyl adduct. Fig. 4 summarizes the in vivo pathways of chlorpyrifos and the following conversion of the adduct into the biomarker of exposure, as result of the sample processing for the diverse analytical methods. Fig. 4 illustrates that DEFP can be formed in the fluoride reactivation assay from four different sources. 4. Conclusion Verification of OP exposure can have several purposes, such as: (i) monitoring health status in order to optimize therapy and (ii) forensic purposes in case of suspected malicious usage of the agent. In addition to the classical methods such as cholinesterase activity, intact agent and metabolites measurements, determination of adducts can have added value. The fluoride reactivation method results in a collection of all fluoride derivatives of the organophosphates from sources varying from the original pesticide to adducts with BuChE, albumin and as yet unknown binding sites, which yields valuable insight into the total body burden of organophosphate anticholinesterases in individuals. LC–MS/MS analysis of adducts to proteins provides a more detailed insight in the origin of these adducts, it is also able to detect the aged OP-adduct which would not be detected with the fluoride reactivation method. Moreover, it helps the interpretation of the results that have been obtained with the fluoride reactivation technique. In this case the formation of DEFP might suggest that the adduct originated from BuChE and would suggest therapy with oximes, to reactivate acetylcholinesterase, might still be effective. However, the analysis of phosphylated BuChE brought up another perspective and revealed that the reactivatable adduct
DEFP was not present on BuChE. In conclusion, the current paper shows that multiple analytical techniques are available for the verification of exposure to an OP pesticide, which enable the retrospective verification of that exposure. In this case the retrospectivity was that long, that even at 49 days after the ingestion of the pesticides, the adducts could still be detected. Therefore these methods can be very useful for research in the field of molecular epidemiology. Finally, the multiple, orthogonal methods pointing unequivocally to a particular exposure, is also highly relevant from a forensic point of view.
Conflict of interest The authors declare that there is no conflict of interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2012.10.014. References [1] F. Eyer, D.M. Roberts, N.A. Buckley, M. Eddleston, H. Thiermann, F. Worek, P. Eyer, Extreme variability in the formation of chlorpyrifos oxon (CPO) in patients poisoned by chlorpyrifos (CPS), Biochem. Pharmacol. 78 (2009) 531– 537. [2] R. Heilmair, F. Eyer, P. Eyer, Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma, Toxicol. Lett. 181 (2008) 19–24. [3] M. Eddleston, P. Eyer, F. Worek, F. Mohamed, L. Senarathna, L. von Meyer, Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study, Lancet 366 (2005) 1452–1459. [4] D.B. Barr, J. Angerer, Potential uses of biomonitoring data: a case study using the organophosphorus pesticides chlorpyrifos and malathion, Environ. Health Perspect. 114 (2006) 1763–1769. [5] M. Polhuijs, J.P. Langenberg, H.P. Benschop, New method for retrospective detection of exposure to organophosphorus anticholinesterases: application to alleged sarin victims of Japanese terrorists, Toxicol. Appl. Pharmacol. 146 (1997) 156–161. [6] C.E.A.M. Degenhardt, K. Pleijsier, M.J. van der Schans, J.P. Langenberg, K.E. Preston, M.I. Solano, V.L. Maggio, J.R. Barr, Improvements of the fluoride reactivation method for the verification of nerve agent exposure, J. Anal. Toxicol. 28 (2004) 364–371. [7] A. Fidder, A.G. Hulst, D. Noort, R. de Ruiter, M.J. van der Schans, H.P. Benschop, J.P. Langenberg, Retrospective detection of exposure to organophosphorus anti-cholinesterases: mass spectrometric analysis of phosphylated human butyrylcholinesterase, Chem. Res. Toxicol. 15 (2002) 582–590. [8] D. Noort, A.G. Hulst, A. van Zuylen, E. van Rijssel, M.J. van der Schans, Covalent binding of organophosphorothioates to albumin: a new perspective for OPpesticide biomonitoring?, Arch Toxicol. 83 (2009) 1031–1036. [9] B. Li, I. Ricordel, L.M. Schopfer, F. Baud, B. Megarbane, F. Nachon, P. Masson, O. Lockridge, Detection of adduct on tyrosine-411 of albumin in humans poisoned by dichlorvos, Toxicol. Sci. 116 (2010) 23–31. [10] B. Li, I. Ricordel, L.M. Schopfer, F. Baud, B. Mégarbane, P. Masson, O. Lockridge, Dichlorvos, chlorpyrifos-oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose, J. Appl. Toxicol. 30 (2010) 559–565. [11] M.J. van der Schans, B.J. Lander, H. van der Wiel, J.P. Langenberg, H.P. Benschop, Toxicokinetics of the nerve agent (+/)-VX in anesthetized and atropinized hairless guinea pigs and marmosets after intravenous and percutaneous administration, Toxicol. Appl. Pharmacol. 191 (2003) 48–62. [12] M.J. van der Schans, A. Fidder, D. van Oeveren, A.G. Hulst, D. Noort, Verification of exposure to cholinesterase inhibitors: generic detection of OPCW Schedule 1 nerve agent adducts to human butyrylcholinesterase, J. Anal. Toxicol. 31 (2008) 125–130. [13] N. Aurbek, H. Thiermann, F. Eyer, P. Eyer, Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: a kinetic analysis, Toxicology 229 (2009) 133– 139.