Biomarkers of Exposure to Organophosphorus Poisons

C H A P T E R

64 Biomarkers of Exposure to Organophosphorus Poisons: A New Motif for Covalent Binding to Tyrosine in Proteins That Have No Active Site Serine Oksana Lockridge, Lawrence M. Schopfer and Patrick Masson

INTRODUCTION Schaffer et al. (1954) reported the astonishing finding that the irreversible inhibition of eel acetylcholinesterase (AChE) by the organophosphorus (OP) agent, diisopropylfluorophosphate (DFP), was the result of covalent binding to serine. In 1959, horse butyrylcholinesterase (BChE) that had been inactivated by treatment with DFP was found to have diisopropylphosphate-labeled serine in the sequence FGESAGAAS (Jansz et al., 1959). Despite this evidence, serine was not immediately accepted as the esteratic site of cholinesterases, because the pKa of serine was so high that significant reaction with OP agents was not expected (Bergmann, 1955). The crystal structures of AChE and BChE confirmed that OP agents make a covalent bond with serine. These structures also provided an explanation for the special reactivity of the active site serine (Sussman et  al., 1991; Nachon et  al., 2005). Nearby histidine and glutamic acid form a catalytic triad with serine that lowers the pKa of the serine, consistent with the expectation that the hydroxy-anion of serine is necessary to make a nucleophilic attack on the phosphorus. The oxyanion hole activates the OP for attack by the serine. The covalent bond between OP and the active site serine of intact cholinesterase is stable, but not irreversible. Hydrolysis can occur with a half-life of between 10 and 35,000  min, depending on the enzyme, OP, Handbook of Toxicology of Chemical Warfare Agents. DOI: http://dx.doi.org/10.1016/B978-0-12-800159-2.00064-6

temperature, pH, and buffer composition. The adduct becomes irreversibly bound to the enzyme after one of the alkyl groups on the OP is lost in a step called aging (Benschop and Keijer, 1966; Michel et  al., 1967). The dealkylated OP makes a stable salt bridge with the protonated histidine of the catalytic triad, so that histidine is no longer available for the dephosphorylation step that would otherwise have restored the enzyme to an uninhibited state. Hundreds of scientists have contributed to this understanding of the mechanism of OP inhibition of AChE and BChE activity. Their studies are the foundation for the use of AChE and BChE as biomarkers of OP exposure. OP-inhibited AChE and BChE are the established biomarkers of OP exposure (Gupta and Milatovic, 2012, 2014). The special features that make them good biomarkers are the following: (i) they react rapidly with OP at low concentrations; (ii) symptoms of acute toxicity always correlate with inhibition of AChE and BChE; (iii) AChE is present in human red blood cells, while BChE is present in human plasma, making it possible to test for OP exposure by measuring enzyme activity in a blood sample; (iv) the OP adduct (nonaged and aged) for common pesticides and nerve agents is relatively stable, making it possible to detect exposure days after the actual event; and (v) the mechanism of irreversible inhibition of AChE and BChE activity by OP is understood.

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USE OF AChE AND BChE BIOMARKERS IN THE CLINIC Most hospitals and forensic laboratories are capable of performing cholinesterase activity assays. Two examples of the usefulness of AChE and BChE activity assays are given next.

Tokyo Subway Attack with Sarin AChE and BChE were useful biomarkers for identifying the poison that intoxicated more than 5000 people in the Tokyo subway. In March 1995, members of the Aum Shinrikyo sect dispersed the nerve agent sarin in trains on the Tokyo subway. Japanese health workers identified the poison as a cholinesterase inhibitor within 2 h of seeing the first patient (Nozaki and Aikawa, 1995). A cholinesterase inhibitor was suspected because victims had physiological signs of cholinergic intoxication, including pinpoint pupils. Laboratory assays showed that red blood cell AChE and plasma BChE activities were inhibited, confirming that the poison was a cholinesterase inhibitor. There are many cholinesterase inhibitors, including OP pesticides and carbamates (CMs). In the case of some OPs, AChE inhibiting compounds are precursor molecules that need bioactivation before they are toxic to humans. The rapid onset of toxic symptoms meant that the agent used in the subway was already activated or a direct AChE inhibitor was involved that needed no activation. The fact that people were becoming intoxicated by breathing the air meant that the poison was volatile. These characteristics suggested that the poison was a nerve agent, very likely sarin. The Forensic Science Laboratory used gas chromatography–mass spectrometry (GC–MS) to identify sarin in crime scene samples (Seto, 2001). Identification of the poison as sarin was crucial to the police in their search for the perpetrators. Years later, new MS methods were developed and used to retroactively identify sarin bound to red blood cell AChE (Nagao et al., 1997) and to plasma BChE from the Tokyo subway victims (Polhuijs et al., 1997; Fidder et al., 2002).

Suicide Attempts Exposures to OP pesticides are declining in the United States following the banning of chlorpyrifos and diazinon for residential use in 2000 (Sudakin and Power, 2007). In 2004, the number of OP exposure cases reported to the American Association of Poison Control Centers (AAPCC) was 7,181, with 4 fatalaties (Bronstein et  al., 2007). In 2012, the number of cases was 3,179, with 1 fatality (Mowry et  al., 2013). The subjects intentionally ingested OP pesticides for the purpose of committing suicide. In contrast, the ingestion of OP pesticides in suicide attempts is a frequent occurrence in rural communities

of Sri Lanka, India, and China, where an estimated 200,000 persons die annually from OP pesticide poisoning (Eddleston and Dawson, 2012). Diagnosis is made on the basis of cholinergic signs of toxicity, smell of pesticides or solvents, and reduced BChE and AChE activity in blood. Patients who survive the suicide attempt are monitored in the hospital for several days; their plasma BChE is assayed daily because recovery of BChE activity is a marker of OP elimination from the body.

METHODS TO DETECT OP ADDUCTS ON AChE AND BChE Cholinesterase Activity Assay Exposure to a toxic dose of OP results in inhibition of AChE and BChE activities. The most common method to measure OP exposure is to assay AChE and BChE activities in blood using a spectrophotometric method (Ellman et al., 1961; Worek et al., 1999; Wilson et al., 2005). The drawbacks of activity assays are that they do not identify the OP agent in question. They show that the poison is a cholinesterase inhibitor, but they do not distinguish between nerve agents, OP pesticides, CM pesticides, and tightly bound, noncovalent inhibitors like tacrine and other anti-Alzheimer drugs. In addition, low-dose exposure, which inhibits less than 20% of the cholinesterase, cannot be determined by measuring AChE and BChE activity because individual variability in activity levels is higher than the percentage of inhibition.

Fluoride Reactivation Followed by GC–MS A new method for identifying exposure to nerve agents was introduced by Polhuijs et  al. (1997). The method is based on the finding that incubation of sarininhibited BChE with 2 M potassium fluoride at pH 4 results in the release of sarin. Sarin is then extracted and analyzed by GC–MS. Polhuijs et  al. (1997) applied their new method to positively identify sarin in serum samples from Japanese victims of the Tokyo subway attack. This method has the advantage that it positively identifies nerve agents and other OPs (van der Schans et al., 2004). The method has been validated by Adams et al. (2004), who found that potassium fluoride released sarin and soman from human BChE, as well as from covalent attachment to human albumin.

Identification of OP-BChE Adducts by Electrospray–Ionization Tandem MS In clinical diagnosis of OP exposure, the tissue most readily available for study is blood. OP adducts of BChE are better candidates for study than OP adducts on

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Methods to Detect OP Adducts on AChE and BChE

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FIGURE 64.1  MS/MS spectrum of the active site peptide of human AChE, covalently bound to tabun and dealkylated by aging or by acid. The identical modified peptide is derived from human BChE treated with tabun, chlorpyrifos oxon, paraoxon, or diazoxon. The ∆ symbol indicates ions that have lost the OP and a molecule of water, converting serine to dehydroalanine. The peak height for 778.2 m/z was 2.5-fold higher than indicated; it was shortened to show the less intense ions.

AChE for the following reasons. Human blood contains 5 mg of BChE and 0.5 mg of AChE per liter. The BChE is in plasma, whereas the AChE is bound to the membranes of red and white blood cells. Most OPs, with the exception of chemical warfare nerve agents, react more rapidly with BChE than with AChE. Fidder et al. (2002) introduced an electrospray–ionization tandem MS method for diagnosing OP exposure by measuring the mass of the OP-labeled active site peptide of human BChE. His starting material was 0.5 mL of human plasma from a victim of the Tokyo subway attack. The mass of the active site peptide was higher by 120 atomic mass units than the mass of the unlabeled active site peptide. This added mass was exactly what was expected from sarin. The peptide’s MS/MS fragmentation spectrum yielded the sequence of the peptide and verified that the OP label was on serine 198, the active site serine. Two important technical details described by Fidder et  al. (2002) are the advantage of digesting with pepsin rather than trypsin, and the fact that during collision-induced dissociation, the OP-labeled serine decomposes to dehydroalanine, with loss of the OP plus a molecule of water. Pepsin digestion of BChE is preferred because pepsin yields a 9-residue active site peptide FGES198AGAAS, whereas trypsin digestion yields a 29-residue peptide SVTLFGES198AGAASVSLHLLSPGSHSLFTR. Short peptides give a more intense signal in the mass spectrometer than long peptides. Figure 64.1 shows the MS/MS spectrum of the active site peptide FGESAGAAS, modified on serine 198 by monoethoxyphosphate. The 904.3 m/z parent ion is particularly interesting because it represents the deamidated (aged) tabun adduct, as well as the dealkylated (aged) chlorpyrifos oxon adduct, and

FIGURE 64.2  Aging or exposure to acid during pepsin digestion results in deamidation of the initial tabun adduct. Aging dealkylates the initial chlorpyrifos oxon adduct. Pepsin digestion yields the identical 904.3 m/z FGESAGAAS peptide covalently modified by mono­ ethoxyphospate from human AChE, human BChE, and horse BChE inhibited by tabun, chlorpyrifos oxon, diethyl paraoxon, diazoxon, or other diethyxophosphate pesticides.

adducts with other common activated pesticides, including diethyl paraoxon and diazoxon (Figure 64.2). Thus, exposure to the nerve agent tabun yields an adduct that is indistinguishable from exposure to common pesticides. Another notable feature of the peptide in Figure 64.1 is that pepsin digestion of human AChE (Swiss protein # P22303), human BChE (Swiss protein # P06276), and horse BChE (NCBI # gi 7381418) yields the same FGESAGAAS active site peptide.

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FIGURE 64.3  OP-serine loses the OP (soman in this case) plus a molecule of water when the peptide is fragmented in the mass spectrometer. This beta-elimination reaction converts OP-serine to dehydroalanine.

The acid condition for pepsin digestion causes the phosphorus–nitrogen bond to break in adducts with tabun and iso-OMPA (tetraisopropyl pyrophosphoramide). This can be used to distinguish between the reactions with Sp and Rp stereoisomers of tabun (Jiang et al., 2013a). Mass spectrometers convert a majority of the OP-labeled serine to dehydroalanine during acquisition of MS/MS spectra. For example, all ions labeled with the ∆ symbol in Figure 64.1 have dehydroalanine in place of OP-serine. Investigators who are not aware of this gas phase chemistry have a difficult time interpreting their MS/MS spectra. The conversion of OP-serine to dehydroalanine is called beta-elimination. The betaelimination reaction is well documented for phosphoserine peptides (Tholey et  al., 1999). Figure 64.3 shows a schematic diagram of the beta-elimination reaction for soman-labeled serine in human BChE. The ease with which the OP-serine bond breaks during collisioninduced dissociation is indicated by the fact that the most intense ion in Figure 64.1 is the 778.2 m/z ion. The peptide at 778.2 m/z is the parent ion that has lost the OP and 1 molecule of water. However, serine is not restored. A molecule of water is abstracted along with the OP, converting the active site serine to dehydroalanine. Fidder et  al. (2002) showed that MS could detect adducts on human BChE with sarin, soman, dimethyl paraoxon, diethyl paraoxon, and pyridostigmine when purified BChE was treated with these poisons. Studies from other laboratories confirmed that OP-BChE adducts could be identified by MS (Tsuge and Seto, 2006; Sun and Lynn, 2007; Li et al., 2008b).

Single-Step Purification of BChE from Human Plasma A technical difficulty with MS of OP adducts on BChE, in a clinical setting, is the need to enrich plasma samples for BChE to make it possible to detect the labeled peptide. Previously, the BChE protein was enriched from plasma by affinity chromatography on procainamide Sepharose (Fidder et al., 2002; Li et al., 2009b; Liyasova

FIGURE 64.4  SDS gel stained with Coomassie Blue shows BChE purified by binding to immobilized monoclonal mAb2. The monoclonal was crosslinked to CNBr-Sepharose. About 98% of the BChE in 0.5 mL of plasma bound to 0.04 mL of beads. The BChE was released from beads with 0.4 M acetic acid. The band at 85 kDa is the BChE monomer, while the band at 170 kDa is the BChE dimer. Immunoglobulin heavy (50 kDa) and light (25 kDa) chains are major contaminants.

et  al., 2011; van der Schans et  al., 2013). In this case, the level of purity was inadequate when procainamide affinity chromatography was the only enrichment step. We had to further enrich by using gel electrophoresis or ion exchange chromatography, or by selectively extracting the OP-labeled active site peptide on titanium oxide microcolumns (Jiang et  al., 2013c). A big advance was the introduction of a single-step purification method by binding to an immobilized monoclonal (Sporty et  al., 2010). The commercially available monoclonal 3E8 (HAH 002-01-02 from Thermo Scientific Pierce) selectively extracts BChE from human plasma when the monoclonal is immobilized on Dynabeads Protein G (Sporty et al., 2010; Carter et al., 2013) or on Dynabeads epoxy (Marsillach et al., 2011). Two other monoclonals, mAb2 (created in the laboratory of Jacques Grassi) and B2 18-5 (created in the laboratory of Steven Brimijoin), also immunopurify BChE from plasma in a single step (Schopfer et  al., 2014). BChE is released from the antibody with acetic acid. Figure 64.4 is a Coomassie-stained gel showing the effectiveness of immunopurification of BChE from plasma in a single step. A band at 85 kDa for the BChE monomer is visible. The major contaminating proteins are immunoglobulins. Immunopurified BChE released with acid is suitable for MS analysis of adducts, but it is not suitable for applications that require active native BChE enzymes because acid denatures the BChE protein.

VII.  ANALYTICAL METHODS, BIOSENSORS AND BIOMARKERS

Why Are New Biomarkers Needed?

WHY ARE NEW BIOMARKERS NEEDED? Not All OPs Inhibit AChE Tri-o-cresyl-phosphate (TOCP), the contaminant in a homemade liquor called “Ginger Jake,” which is responsible for delayed neuropathy and paralysis of the legs, is bioactivated in a form that inhibits neuropathy target esterase (NTE) and BChE, but not AChE at nonlethal doses (Casida and Quistad, 2004; Glynn, 2006). Large structures with a 12–20 carbon alkyl chain on the phosphorus atom inhibit fatty acid amide hydrolase but not AChE (Casida and Quistad, 2004). These examples show that OPs that do not affect AChE can react with other proteins, sometimes generating toxic symptoms. These examples also suggest that additional, unknown OP targets may exist that are sensitive to OP that do not affect cholinesterases. In order to detect exposure to such OPs, biomarkers other than cholinesterases are needed.

OP doses too Low to Inhibit AChE Cause Toxicity Workers in India (n = 59) engaged in the manufacture of quinalphos had normal red cell AChE activity but complained of generalized weakness and fatigue. They had significantly low scores for memory, learning ability, and vigilance compared to controls. Plantar reflexes were abnormal in 50% of the workers. The average age of the workers was 30 ± 6 years, and the average duration of exposure to quinalphos was 5.7 years. There was no history of acute poisoning. The plant was situated in large tin sheds without adequate ventilation (Srivastava et al., 2000). The most logical explanation for their symptoms was low-dose exposure to OP. Family tobacco farmers in Brazil (n = 37) used chlorpyrifos and acephate for 3 months a year, for 5.4 h a day. The average duration of exposure was 18 years. Their plasma cholinesterase activity was within the normal range and was not different between on- and off-exposure periods. Clinically significant extrapyramidal symptoms were present in 12 subjects during the pesticide application season, though this number was reduced to 9 after 3 months without exposure. Generalized anxiety disorder was diagnosed in 13 subjects, and major depression in 8 subjects. After 3 months without OP exposure, the number of subjects with psychiatric disorders declined to about half (Salvi et al., 2003). The most logical explanation for their symptoms was low-dose exposure to OP. More examples of chronic low dose, subclinical exposure to OPs in humans, leading to chronic neurotoxicity, are cited in a number of studies (e.g., Abou-Donia, 2003; Kamel and Hoppin, 2004). Animal studies have been directed at understanding the mechanism of low-dose OP toxicity. The studies agree

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that doses too low to inhibit AChE activity nevertheless have adverse effects on the animals, including disruption of adenylyl cyclase signaling (Song et  al., 1997), hyperphosphorylation of calcium/cyclic adenosine monophosphate (cAMP) response element binding protein (Schuh et al., 2002), airway hyperactivity (Lein and Fryer, 2005), changes in expression levels of fibroblast growth factor in the brain (Slotkin et al., 2007), changes in serotonin receptors (Slotkin et al., 2008), inhibition of acylpeptide hydrolase (Richards et al., 2000), and disruption in lipid metabolism (Medina-Cleghorn et al., 2013). Rats chronically treated with low doses of chlorpyrifos or DFP have long-term cognitive deficits in the absence of clinical signs of exposure (Jett et al., 2001; Terry et al., 2007, 2014). The mechanism for impairment of cognitive function may involve disruption of the microtubule transport of vesicles, organelles, and other cellular components that are synthesized in the neuronal cell body and moved down long axons to presynaptic sites (Gearhart et al., 2007). A possible, noncholinesterase-based explanation for the neurotoxic symptoms observed in humans from the previous examples is that workers were also exposed to a combination of chemicals, including heavy metals, solvents, herbicides, and fumigants. The symptoms may actually have been caused by these chemicals and not by the OP (Kamel and Hoppin, 2004). However, the animal data cannot be explained this way because the animals were treated only with OP agents. In conclusion, OP targets that are not AChE or BChE are involved in chronic neurotoxicity. These unknown targets bind OP at doses too low to inhibit AChE.

Only Some People Have Symptoms Not every person who has been chronically exposed to OP exhibits symptoms. For example, of 612 sheep dippers (workers who dip sheep into a chemical bath to kill insects in the wool) exposed to diazinon twice a year for 3 days at a time for 40 years, only 19% reported symptoms. In a control group of ceramics workers who had not been exposed to OPs, 5% reported similar symptoms (Pilkington et al., 2001). The susceptibility of a minority of the population may be explained by variations in genes affecting OP metabolism. The best-studied example is paraoxonase, an enzyme that inactivates OPs (La Du et al., 2001; Furlong, 2007; Costa et al., 2013). Paraoxonase polymorphism in humans is proposed to explain why some people are resistant to OP toxicity, while others are susceptible. Another enzyme that may be involved in resistance to OP toxicity is BChE. BChE scavenges OPs, eliminating the poison before it reaches sites where it could cause harm (Doctor et al., 1991). Humans have a wide range of BChE activities, with some people having none whatsoever due to genetic variation (Manoharan et al., 2007). It is possible,

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but unproven, that people with BChE deficiency are more susceptible to OP toxicity. Polymorphisms in liver cytochrome P450 enzymes may also contribute to individual susceptibility to OP toxicity. Specific cytochrome P450 enzymes bioactivate the parent organophosphorothioates to the highly toxic oxon forms. Other cytochrome P450 enzymes detoxify OPs by dearylation (Hodgson and Rose, 2007). The concentration of each isozyme varies among individuals. Thus, the relative rates for activation and detoxification and the circulating levels of the toxic form of the OP will vary between individuals. Another possibility for variable susceptibility in the population could be genetic variation in the yetunknown OP targets that are responsible for the symptoms. Some forms of these targets may be more sensitive to OPs than are others.

Toxic Symptoms Depend on the OP High doses of OPs cause similar toxic effects, independent of the identity of the OPs. However, low-dose effects are not identical for all OPs (Moser, 1995). For example, a low dose of fenthion decreased motor activity in rats by 86% but did not alter the tail-pinch response, whereas a low dose of parathion did not affect motor activity but did decrease the tail-pinch response. In another example, rats given doses of different OPs that inhibited AChE to similar levels had more severe toxicity when the OP was parathion than when it was chlorpyrifos (Pope, 1999). Toxicological studies such as these have led to the conclusion that sites in addition to cholinesterase are targets of OPs. New biomarkers for these effects of OPs are needed. In summary, AChE and BChE are not the only proteins modified by OP exposure in humans. Neurotoxicity from low doses of OPs may be explained by OP modification of heretofore unidentified proteins. Toxic symptoms from low-dose exposure to a particular OP are not identical to toxic symptoms from another OP, suggesting that the set of proteins modified by a particular OP does not overlap completely with the set of proteins modified by a different OP. Identification of new biomarkers of OP exposure could lead to new assays for OP exposure, and could lead to an understanding of the causes of low-dose toxicity.

NEW BIOMARKERS Beta-Glucuronidase in Rat Plasma A carboxylesterase in rat liver microsomes called egasyn is tightly linked to beta-glucuronidase (BG) by a complex. When egasyn binds OP, it releases BG into the blood. A single oral dose of chlorpyrifos (10 mg/ kg) increased the level of BG activity in rat blood 100fold within 2 h (Fujikawa et  al., 2005). The BG activity

decreased to control levels by 24 h. Thus, increased levels of BG in plasma may serve as a biomarker for OP exposure. However, increase in plasma BG activity has not been validated in other animal species or in humans. For further details on BG, refer to Satoh et al. (2010).

Acylpeptide Hydrolase in Rat Brain Acylpeptide hydrolase is a member of the serine hydrolase family. It deacetylates the acetylated N-terminus of polypeptides. Rat brain acylpeptide hydrolase was inhibited 93% at a dose of dichlorvos (4 mg/kg, i.p.) which inhibited AChE only 47%. The in vitro sensitivity of acylpeptide hydrolase to chlorpyrifosmethyl oxon, dichlorvos, and DFP (IC50) was 6–10 times greater than that of AChE (Richards et al., 2000). Acylpeptide hydrolase is also found in human erythrocytes, where it could potentially serve as a biomarker for low-dose exposure to OP in humans (Quistad et al., 2005), though human cases of OP exposure have not yet been tested for OP-modified acylpeptide hydrolase.

Albumin in Mouse and Guinea Pig Plasma Mice treated with a nontoxic dose of a biotin-tagged OP called fluorophosphate (FP)–biotin had FP-biotinylated albumin in blood and muscle (Peeples et  al., 2005). In vitro experiments identified the site in human albumin for covalent attachment of a variety of OPs as tyrosine 411 (Li et al., 2007). Guinea pigs treated with the nerve agents soman, sarin, cyclosarin, or tabun had nerve agent-labeled albumin in their blood (Williams et al., 2007). The OPs were bound to tyrosine. The tabun-tyrosine and soman-tyrosine adducts were detected in blood 7 days postexposure, indicating that the adducts are stable. The adducts did not undergo aging and were not released from tyrosine by treatment of the guinea pigs with oxime, which is a common treatment for OP exposure that induces release of OP from AChE. MS identified OP-albumin adducts in rats treated with 1/5 LD50 of paraoxon (Gladilovich et al., 2010). These examples show that OPs can bind covalently to albumin under physiological conditions, and that the resultant adducts are relatively stable. OP-albumin adducts could therefore be useful as biomarkers of OP exposure. In addition, unlike cholinesterases, the soman-albumin conjugate does not age (Li et al., 2008a), making it possible to discriminate between sarin and soman exposure.

Albumin in Human Plasma is a Biomarker of OP Exposure The animal studies described previously provided the rationale for testing whether humans poisoned by OP have detectable OP-albumin adducts. To date, three

VII.  ANALYTICAL METHODS, BIOSENSORS AND BIOMARKERS

Motif for OP Binding to Tyrosine

studies provide MS evidence that plasma from humans who ingested dichlorvos (Li et  al., 2010b) or chlorpyrifos (Li et  al., 2013b; van der Schans et  al., 2013) contains OP-albumin adducts. Blood drawn as late as 49 days after exposure had detectable levels of OP adducts on tyrosine (van der Schans et  al., 2013). Though the chlorpyrifos-poisoned patients had been treated with oximes to reverse OP binding to AChE, their albumin had detectable levels of OP adducts. This suggests that oximes do not reverse OP binding to albumin.

M2 Muscarinic Receptors in Heart and Lung The 3H-chlorpyrifos oxon binds covalently to rat heart M2 muscarinic receptors (Bomser and Casida, 2001). The site of attachment has not been identified. When guinea pigs were treated with chlorpyrifos, diazinon, or parathion at doses too low to inhibit AChE activity, the M2 muscarinic receptors lost their ability to inhibit acetylcholine release from parasympathetic nerves, causing bronchoconstriction (Lein and Fryer, 2005).

COVALENT BINDING OF OP TO TYROSINE In a 1963 Pedler lecture, Sanger reported that 3HDFP makes a covalent bond with tyrosine in the sequence ArgTyrThrLys from human and rabbit albumin (Sanger, 1963). MS of human albumin treated with soman, chlorpyrifos oxon, FP-biotin, dichlorvos, and DFP confirmed OP modification on Tyr 411 in peptide LVry*tkKVPQVSTPTL (Li et al., 2007, 2008a), where the lowercase letters show Sanger’s sequence and the asterisk indicates the labeled tyrosine. At the time that we confirmed Sanger’s observations, we thought that albumin was a special case, though we were aware that papain and bromelain had also been reported to bind 3H-DFP on tyrosine (Murachi et  al., 1965; Chaiken and Smith, 1969). We soon learned differently. In a general search for proteins that bind OP, we treated live mice and mouse tissues with FP-biotin, a modified OP designed for identifying unknown targets of OP reaction. The FP-biotinylated proteins were isolated on immobilized avidin, washed with 0.1% sodium dodecyl sulfate (SDS), and separated on an SDS gel. Coomassie Blue-stained bands were excised and digested with trypsin and the proteins in the bands were identified by MS. Surprisingly, the majority of proteins that were identified were not serine esterases or proteases, which are the classical targets for OPs, nor did the identified proteins contain the consensus sequence GXSXG, which is characteristic of an active site serine. Typically, we found high-abundance proteins such as albumin and tubulin.

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Though biotin–avidin complexation has been used widely to isolate specific targets, it is notorious for generating false-positive identifications. To rule out the possibility that our results were an artifact, we set out to find the OP-labeled peptide. We reasoned that convincing proof for OP labeling required identification of the labeled peptide and the labeled amino acid. During years of MS analysis, we had consistently identified OP-labeled tubulin in mouse brain. Therefore, we studied pure bovine tubulin (Cytoskeleton, Inc., Denver, CO) by treating it with soman, sarin, FP-biotin, DFP, chlorpyrifos oxon, and dichlorvos. We isolated the OP-labeled tryptic peptides and analyzed them by fragmentation in the QTRAP 2000 and QTRAP 4000 mass spectrometers. We identified five OP-labeled peptides in tubulin (Grigoryan et al., 2008). In every peptide, the OP was covalently attached to tyrosine. Similar MS experiments with pure human and mouse transferrin (Li et al., 2009a), and with human kinesin showed that the OP label was consistently on tyrosine. Studies with human plasma identified OP labeling on tyrosine in apolipoprotein and alpha-2-glycoprotein. Aggressive treatment of human albumin with FP-biotin and chlorpyrifos oxon led to the identification of seven OP-labeled tyrosines (Ding et al., 2008). Finally, we found that small synthetic peptides made a covalent bond with DFP, chlorpyrifos oxon, dichlorvos, and soman (Li et al., 2009a). For example, incubation of peptide RYGRK (ArgTyrGlyArgLys) with soman yielded the pinacolylphosphonate-modified peptide (Li et al., 2013a). MS analysis conclusively proved that the OP was attached to tyrosine.

MOTIF FOR OP BINDING TO TYROSINE Comparison of the sequences of OP-labeled peptides shows no consensus sequence around the tyrosine to which the OP binds. What the peptides do have in common is the presence of a positively charged arginine, lysine, or histidine within five amino acids of the labeled tyrosine, most being within three amino acids. We suggest that these positively charged residues could interact with the phenolic hydroxyl of tyrosine to lower the pKa. Such ion-pair interactions have been shown to lower the pKa for the negatively charged partner, with a comparable rise of the pKa of the positively charged partner by as much as 4 pKa units (Johnson et al., 1981). Tyrosines with a lower pKa value would be better nucleophiles and thus better able to attack OPs. In conclusion, most proteins that we have examined in detail using sensitive MS techniques have shown the capacity to become labeled by OP on tyrosine. However, only certain tyrosines in a protein are labeled (i.e., those on the surface and near a positively charged residue that

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could potentially activate the phenolic hydroxyl group). The finding that even small synthetic peptides can be labeled by OP on tyrosine has led to the hypothesis that OP labeling on tyrosine is a general phenomenon. We propose that OP labeling on tyrosine is a new motif for OP binding to proteins.

CHARACTERISTICS OF OP BINDING TO TYROSINE On-Rate Little is known about the rate of OP binding to tyrosine because the recognition of this OP binding motif is new. Soman binding to Tyr 411 in human albumin has been measured and has been found to be slow, with a bimolecular rate constant of 15 ± 3 M−1 min−1 (Li et al., 2008a). We expect that other proteins will be identified whose rate of OP binding to tyrosine will be faster.

Off-Rate The OP adduct on tyrosine 411 of human albumin is stable. The half-life for decay of the soman–Tyr 411 adduct is 20 days at pH 7.4, 22°C (Li et al., 2008a). The chlorpyrifos oxon–Tyr 411 adduct is even more stable. After 7 months at 22°C in pH 7.4 buffer, 80% of the Tyr 411 was still labeled with diethoxyphosphate, which is the adduct formed by chlorpyrifos oxon. However, at pH 8.3 and 22°C, 50% had lost the OP label in 3.6 months. OP-albumin adducts stored at −80°C are stable indefinitely at pH 8.3, 7.4, and 1.5. One advantage of a stable OP-tyrosine adduct is that it will survive in an animal long enough to allow the generation of antibodies. Another advantage of a stable OP-tyrosine adduct is that detection of OP can be made on samples long after exposure.

No Aging OPs bound to tyrosine do not age. Aging of OP adducts on AChE and BChE is defined as the loss of an alkoxy group from the phosphorus atom. No masses representing aged OP-tyrosine adducts have been found. Aging can confound the identification of the original OP. For example, aged sarin and soman adducts on AChE are indistinguishable because they yield the same methylphosphonate derivative. The absence of aging for OP-tyrosine adducts results in a species that is suitable for discriminating between sarin and soman adducts because the unaged adducts have different masses. Absence of aging for OP-tyrosine adducts has also been reported for albumin adducts in guinea pigs (Williams et al., 2007).

METHODS FOR DETECTING OP BINDING TO TYROSINE MS for OP Adducts on Unknown Proteins If the identity of the OP-labeled protein is unknown, a tagged OP (e.g., a biotinylated OP) can be used to identify the protein (Schopfer et  al., 2005). After the identity of the OP-labeled protein is known, identification of the OP-labeled peptide depends on separating it from contaminating peptides. We have found that the OP-labeled peptide is frequently not found by MS unless it has been extensively purified. In some cases, it is possible to identify the labeled peptide simply by liquid chromatography–tandem MS (LC-MS/MS), where an enzymatic digest of the isolated protein is subjected to liquid chromatography (LC) on a C18 nanocolumn, and the effluent from the column is electrosprayed directly into the mass spectrometer. For other cases, offline highpressure liquid chromatography (HPLC) purification of the enzymatic digest is necessary to obtain a purified fraction of peptides that can be introduced into the mass spectrometer. Preliminary identification of the labeled peptides in HPLC fractions is made by mass, using matrix-assisted laser desorption/ionization (MALDI) MS (MALDI TOF– TOF 4800 from Applied Biosystems). The MS-Digest algorithm, available from the Protein Prospector website at the University of California, San Francisco (prospector.ucsf.edu/prospector/mshome.htm), is a useful tool for predicting the masses of peptides from a proteolytic digest both with and without the added mass from a particular OP. Another indispensable tool is the Fragment Ion Calculator (available from http:// db.systemsbiology.net:8080/proteomicsToolkit/), which can be used to calculate the masses of ions generated during MS/MS fragmentation of a peptide. Our laboratory acquired MS/MS spectra with the MALDI TOF– TOF 4800, the QTRAP 4000, and Triple TOF 5600 mass spectrometers (AB-Sciex). Examples of MS/MS spectra for human albumin peptides labeled on tyrosine with chlorpyrifos oxon are shown in Figure 64.5.

Fluoride Treatment to Release OP from Albumin The fluoride reactivation method has been applied to a human case of self-poisoning by chlorpyrifos (van der Schans et  al., 2013). Blood drawn from the patient 49 days after she ingested chlorpyrifos yielded two products, both released from albumin with potassium fluoride. The released products were identified by LC-MS using multiple reaction monitoring (MRM). The products were diethoxyfluorophosphate (representing an albumin adduct with chlorpyrifos oxon) and

VII.  ANALYTICAL METHODS, BIOSENSORS AND BIOMARKERS

FIGURE 64.5  MS/MS spectra of human albumin peptides labeled on tyrosine with chlorpyrifos oxon. (A) The doubly charged parent ion (617.7 amu) for KYLYEIAR includes a mass of 136 amu from chlorpyrifos oxon and a mass of 43 amu from CM. Carbamylation was a by-product of denaturation in 8 M urea. The presence of diethoxyphosphate on Tyr 138 is supported by the masses y7, a2—amine (17 amu), b2—amine (17 amu), b3—water (18 amu), a4—amine (17 amu), and b5—amine (17 amu), all of which include the 136 amu added mass for diethoxyphosphate. The ion at 272.4 amu is consistent with the mass of the diethoxyphosphotyrosine immonium ion; its presence supports chlorpyrifos oxon labeling of tyrosine. (B) The singly charged parent ion (547.2 amu) for YTK includes an added mass of 136 amu from chlorpyrifos oxon. The presence of diethoxyphosphate on Tyr 411 is supported by the masses of the b1 ion, the y2 ion, and the parent ion, all of which include the 136-amu mass of diethoxyphosphate. Furthermore, the characteristic ions at 272.2 amu for the diethoxyphosphotyrosine immonium ion, at 328.2 amu for the monoethoxyphosphoTyrThr ion minus amine (17 amu), and at 244.2 amu for phosphotyrosine support labeling on tyrosine. The loss of one or both alkyl groups from the diethoxyphospho adduct during collision-induced dissociation in the mass spectrometer is a common observation. Source: Reprinted with permission from Ding et al. (2008). Copyright 2008 American Chemical Society.

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diethoxyfluorothiophosphate (representing an albumin adduct with the thiol form of chlorpyrifos). It is remarkable that the albumin adducts were still detectable 49 days after exposure, when the patient had recovered from the toxic effects.

MRM LC-MS/MS for OP Adducts When the Protein and the Modified Amino Acid Are Known The most sensitive LC-MS/MS method for detection of OP exposure is a method called multiple reaction monitoring or selective ion monitoring. This method requires knowledge of the mass of the parent ion and masses of two or more product ions. The selection of product ions is determined experimentally with known samples because not all product ions give a good signal. The method is sensitive because the mass spectrometer selects only those ions that fit the given parent and product ions. An MS/MS spectrum is automatically acquired for samples that have the specified parent and product ions. The MS/MS spectrum allows the investigator to determine whether the ion is the OP peptide or whether it has the specified masses by coincidence. A list of the parent ion and product masses for nerve agent tyrosine adducts is published (Williams et al., 2007). The MRM method has been used to detect nerve agent–tyrosine adducts in the blood of guinea pigs (Williams et al., 2007), carbofuran-BChE adducts in the blood of human subjects (Li et  al., 2009b), OP-BChE adducts in the blood of humans poisoned by dichlorvos, chlorpyrifos, and aldicarb (Li et al., 2010a), OP-albumin adducts in the blood of humans poisoned by dichlorvos (Li et  al., 2010b), nerve agent–tyrosine and nerve agent–BChE adducts in the blood of marmosets (Read et  al., 2010), nerve agent–tyrosine in rat plasma (Bao et  al., 2012), and OP-tyrosine adducts in the plasma of human subjects poisoned by chlorpyrifos (van der Schans et al., 2013). The MRM method is sensitive and quantitative, but it has the disadvantage that unexpected adducts are not found. The investigator will find only known adducts. A new poison is likely to make adducts that are undetectable in an MRM method.

Pronase Digestion to Yield Single Amino Acids Modified by OPs Digestion of OP-treated human plasma proteins with pronase yields free amino acids modified by OP (Black et  al., 1999). Human plasma treated with sarin or soman contains OP-serine (from BChE) and substantially greater amounts of OP-tyrosine (from albumin and possibly other proteins as well; Black et  al., 1999).

This method can detect low levels of exposure because large volumes of plasma can be digested. The Porton Down laboratory has synthesized isotope-labeled internal standards (Williams et al., 2007). MRM methods have been published for the detection of nerve agent–tyrosine adducts (Williams et al., 2007; Bao et al., 2012). Less than 1% of the albumin in plasma is modified by OP. A cleanup step is required that separates and concentrates the OP-tyrosine adduct, such as solid phase extraction (Williams et  al., 2007; Read et  al., 2010) or offline HPLC (Jiang et al., 2012), before the sample is analyzed by LC-MS/MS. However, the use of MRM in an LC-MS/ MS protocol increases the sensitivity and specificity of detection, making it possible to identify OP-tyrosine in a pronase digest directly, without a cleanup step (van der Schans et al., 2013).

Enrichment of OP-Albumin Pepsin Peptides on PHOS-Select Iron Affinity Beads Though several tyrosine residues on albumin can be modified by treatment with OP, the most reactive is tyrosine 411. Peptides VRY411TKKVPQVST, and LVRY411TKKVPQVST selectively bind to Fe(3+) beads at pH 11. Human plasma digested with pepsin and diluted with buffer to raise the pH to 11 is applied to a microcolumn of beads. The peptides are eluted with pH 2.6 buffer and identified by MS. The protocol enriches both the unmodified and OP-modified peptides, regardless of the identity of the OP, thus allowing quantitation of the OP-peptide relative to the unmodified peptide (Jiang et al., 2013b).

Antibody Sensors that use an antibody to detect nerve agent– adducts on albumin are being developed but are not yet commercially available (Vandine et al., 2013).

OPS MAKE A COVALENT BOND WITH SERINE, THREONINE, TYROSINE, LYSINE, AND HISTIDINE The first OP adducts were identified on the active site serine of AChE and BChE using a radioisotope-labeled OP. For 50 years, it was taken as dogma that an enzyme was defined as a serine hydrolase if it made a covalent bond with an OP. When a denatured protein sample made a covalent bond with an OP, the result was discarded as an outlier. Today, it is known that proteins need not be in their native structure to bind OPs. Small peptides make a covalent bond with OPs. The new tools of MS have demonstrated that proteins are modified

VII.  ANALYTICAL METHODS, BIOSENSORS AND BIOMARKERS

REFERENCES

by OPs not only on serine, but also on threonine, tyrosine, lysine, and histidine (Grigoryan et  al., 2008, 2009; Liyasova et  al., 2012; Verstappen et  al., 2012). To date, these additional binding sites have been demonstrated by in vitro reactions with high concentrations of soman, chlorpyrifos oxon, FP-biotin, dichlorvos, and cresyl saligenin phosphate, but it seems only a matter of time before new protein targets will be discovered that react with low doses of OP.

CONCLUDING REMARKS AND FUTURE DIRECTIONS A new motif for OP binding to tyrosine has been identified. Almost all proteins appear to be capable of binding OP covalently on tyrosine. Whether or not OP will bind to tyrosine in vivo depends on the concentration of the protein, the concentration of the OP, and the ionization status of the tyrosine hydroxyl group. The latter factor appears to depend on the presence of nearby positively charged residues. Albumin is the first protein outside the serine hydrolase family to be recognized as a target of OP binding in humans. The concentration of albumin is so high (600 μM in human plasma) that albumin binds OP despite its slow rate of reaction. The reaction of albumin’s most sensitive tyrosine is aided by the presence of three positively charged residues within a five-residue stretch surrounding that tyrosine. It is expected that in the future, additional proteins will be identified as targets of OP binding. Antibodies will be used to diagnose OP exposure in a biosensor assay with blood, saliva, sweat, or urine. New biomarkers of OP exposure will be identified using MS and antibodies. The identification of new biomarkers for low-dose OP exposure is expected to lead to an understanding of how neurotoxicity is caused by OP doses that are too low to inhibit AChE. For example, it is possible that disruption of microtubule polymerization by OP-adduct formation may explain cognitive impairment from OP exposure. Identification of new biomarkers of OP exposure may also lead to an understanding of why some people are intoxicated by low doses of OP that have no effect on the majority of the population. The new motif for OP binding to tyrosine may lead to new antidotes for OP poisoning; for example, peptides containing several tyrosines and several arginines may be effective OP scavengers.

Acknowledgments Mass spectra were obtained with the support of the Mass Spectrometry and Proteomics core facility at the University of Nebraska Medical Center. This work was supported NIH Eppley Cancer Center grant P30CA36727, and DGA grant 03co010-05/PEA01 08 7 (to P.M.).

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VII.  ANALYTICAL METHODS, BIOSENSORS AND BIOMARKERS