Dr Daric Erminote

Jan 12, 2021

45 min read

Biological Hazard Markers of Exposure to Chemical Welfare Agents and Analytics Method

The first Gulf war increased the attention given to cyclosarin (GF), previously considered a nerve agent of secondary importance, while further development work has been done on newer oxime nerve agent treatments, such as HI-6. In addition, a considerable amount of work has been carried out on the skin effects of sulphur mustard. The terrorist incidents using nerve agents, which took place in 1994 and 1995 in Japan, kindled a considerable amount of interest in other countries and gave rise to a number of symposia, such as the seminar on responding to the consequences of chemical and biological terrorism held at Bethesda, Maryland, in July 1995. Subsequent events, such as the 9/11 attacks in New York and Washington and the Bali, Madrid and London tube bombings, none of which involved chemical agents, have increased the attention given to the possibility of the use of chemicals by Al-Qaida and other groups. The possibility of the terrorist use of chemical weapons means that the management of civilian casualties has to be considered. Previously, management of chemical casualties has generally been in the context of military personnel, who may be protected physically and in some cases, by pharmacological preparations, against chemical warfare agents, and who will in any case usually be young and physically fit. Civilian casualties, by contrast, may include the infirm, the elderly and children. In addition, armed forces may have in place procedures for dealing with chemical attacks, whereas, until recently, that was not the case for civilians. Most western countries now have in place some procedures to deal with civilian casualties in the event of a terrorist attack using chemicals. However, many problems remain, including, for example, the need for mass decontamination after an incident. This and other topics receive special attention. It is now about ninety years since chemical weapons were used on a large scale during World War I. That these weapons still pose a threat to both civilians and military personnel says little for mankind’s socio-political progress. We hope this may stand as a small memorial to his work in this area.

Disclaimer: The use of chemical weapons is prohibited by a number of international treaties or conventions. This illegality is said to confirm and codify a customary prohibition based on concepts of ethics and morals. Despite these prohibitions, and unlike other weapon systems also prohibited, the number of countries which have acquired or which are acquiring chemical weapons continues to increase. It might be argued that this is explained, in part, by the lack of force of international agreements resulting from their being based upon false assertions and their being established largely as a result of vigorous propaganda exercises in the aftermath of a world war. Current international moves to abandon chemical weapons may well be brought to a successful conclusion but, with the lack of a sound and persuasive argument against the use of such weapons, it remains very unwise to assume that clandestine production of such weapons will not be attempted and that they will not be used during a war. In addition, the lack of a sound ethical compulsion to desist from using such weapons may very likely lead to their proliferation and use in the so called Third World conflicts, particularly among nations with no memory of WWI.

Introduction

Biological Reactions of CW Agents

The most abundant free nucleophiles in the body are water and the tripeptide glutathione (γ- Glu — Cys — Gly), which has a reactive thiol group associated with the cysteine residue. Reactions with these nucleophiles, either chemically or me- diated by enzymes, are the starting point for most metabolic pathways of electrophiles and account for most of the metabolites excreted in urine. A host of nucleophilic sites also exist on proteins,

Figure 1. Typical profiles for urinary metabolites and protein adducts in blood in relation to required limits of detection

DNA and other macromolecules (Van Welie et al., 1992). Examples are cysteine (SH), serine and tyrosine (OH), lysine and N -terminal valine (NH2), and aspartic and glutamic acid (CO2H) amino acid residues on proteins, and NH and PO−3 residues on DNA. Haemoglobin and albumin are the most abundant proteins in blood, and covalently bound residues on these proteins provide biological markers of exposure to many electrophiles, including CW agents. Nerve agents are somewhat more selective in that they specifically target a serine OH group in the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Sulphur and nitrogen mustards react with guanine NH residues in DNA, which are present in every tissue in the body.

As described below, urinary metabolites have been identified for vesicants, nerve agents, 3- quinuclidinyl benzilate (BZ), hydrogen cyanide and the RCAs, CS, CR and capsaicin. Protein adducts have been identified for vesicants, nerve agents and phosgene, and DNA adducts for sul- phur and nitrogen mustards. With the rapid ad- vances being made in proteomics and metabo- nomics, new biological markers of exposure will undoubtedly be identified in the near future.

Free Metabolites as Biological Markers

Protein and DNA Adducts as Biological Markers

Haemoglobin and albumin are the two most abundant proteins in blood, and for reactions that are purely chemical (i.e. non-enzymatic) usually provide the most abundant biomarkers. In the case of nerve agents, enzymatic reactions with the enzymes AChE and BuChE provide sensitive biomarkers. Haemoglobin and albumin are water-soluble proteins that have a large number of hydrophilic and nucleophilic amino acid residues on the periphery of the molecule. An important difference is that an agent must penetrate the red cell membrane to react with haemoglobin, and this will depend partially on its physical properties. Albumin has a cysteine residue (34) which is much more accessible than those in haemoglobin. Ideally, blood should be separated into red cell and plasma fractions and stored frozen shortly following collection, but this is not always possible.

Many electrophilic toxicants, including ethy- lene oxide, methyl bromide and acrylamide, react with haemoglobin and albumin (To ̈rnqvist et al., 2002). It is therefore not surprising that one or both of these proteins form adducts with vesicants, nerve agents and phosgene, at least in vitro. Adducts with haemoglobin have been iden- tified for sulphur mustard (Noort et al., 1996; Black et al., 1997b), lewisite (Fidder et al., 2000) and phosgene (Noort et al., 2000). Adducts with albumin have been identified for sulphur mustard (Noort et al., 1999) and nitrogen mustard HN-2 (Noort et al., 2002b), some nerve agents (Black et al., 1999; Harrison et al., 2006) and phosgene (Noort et al., 2000). Adducts with AChE and BuChE have been identified for the range of nerve agents (Fidder et al., 2002; Elhanany et al., 2001). Another possible source of protein adducts is skin. Sulphur mustard forms adducts with keratin aspartic and glutamic acid residues (Van der Schans, G.P. et al., 2003). Hair is also a possible source of protein adducts but, unlike albumin and haemoglobin, most of the nucleophilic sites are on the interior of the protein with hydrophobic residues on the exterior.

DNA adducts provide biomarkers of exposure for sulphur and nitrogen mustards. However, turnover of these adducts appears to be much shorter than the lifetime of natural DNA due to the intervention of repair mechanisms.

Analytical Methods

Mass spectrometry is generally the only spectrometric technique that universally provides the requisite combination of sensitivity and specificity for analysis at low parts per billion (ppb) in urine and blood. Nuclear magnetic resonance (NMR) spectrometry, although a very specific technique, still does not have the sensitivity required for trace analysis in complex matrices. Mass spectrometry is usually used in combina- tion with gas or liquid chromatography, or less commonly with capillary electrophoresis. For analysis down to mid — low ppb levels, single stage GC — MS or LC — MS may provide adequate limits of detection, but for detection at low-sub ppb levels with a high degree of confidence tandem mass spectrometry is required. Immunoassays are useful for the rapid screening of multiple samples but, unless antibodies for the analyte are available, they require a considerable effort to develop.

Most metabolites, particularly those derived from hydrolysis, are polar and require derivatization prior to analysis by GC — MS. A disadvantage of this approach is that derivatization can be a major source of error in trace analysis. The metabolites normally require isolation from aqueous media before derivatization, and this can result in loss of analyte. In addition, the presence of large amounts of extraneous material in an extract, plus residual traces of water, may suppress derivatization. An advantage of derivatization is that it can be used to enhance detection. Conver- sion to perfluorinated derivatives and detection by negative-ion chemical ionization mass spectrometry (NICI-MS) provides the lowest limits of detection for TDG and phosphonic acids. LC — MS (usually LC — MS — MS) provides an alter- native that can be applied directly to concentrated aqueous solutions of metabolites, in most cases without the need to derivatize. LC — MS — MS can provide very low limits of detection, but for many of the simpler polar metabolites, such as TDG and phosphonic acids, lowest limits of detec- tion have been obtained with derivatization and GC — MS — MS. However, LC — MS instrumenta- tion is still improving, particularly with the wider use of capillary columns.

A number of strategies can be applied to the detection of protein adducts. Some proteins can be selectively isolated from blood by affinity SPE; alternatively, blood may be fractionated using precipitation techniques. Detection of the en- tire protein adduct is possible with modern MS techniques, e.g. using electrospray or desorption ionization methods, but limits of detection are usually modest with considerable chemical back- ground. A common approach is to selectively di- gest the protein with enzymes, such as trypsin or pepsin, to produce short-chain peptides, and de- tect the alkylated or phosphylated peptide using LC — MS — MS. Alternatively, the protein may be digested to its constituent amino acids using the protease from Streptomyces griseus (Pronase) or 6 M hydrochloric acid, although these methods tend to produce a large chemical background. In some cases, particularly where ester linkages are formed, the bound moiety may be displaced from the protein by hydrolysis, nucleophiles such as fluoride ion, or chemical derivatization, and detected using a simpler methodology, usually GC — MS.

An important aspect of trace analysis is that rigorous quality control must be included in an- alytical protocols if the results are to withstand international scrutiny. In cases of allegations of CW use, a chain of custody must be maintained and any positive analysis must be preceded by

Figure 2. Sulphur mustard adduct formation with various nucleophiles

a negative control sample taken through the entire analytical procedure to demonstrate that no cross-contamination of equipment has occurred. Acceptable criteria for the trace-level detection of biological markers of CW agent poisoning are currently being discussed with the Organization for the Prohibition of Chemical Weapons.

Sulphuric Mustard

Biological fate

Distribution and Metabolism

The chemical and metabolic reactions of sulphur mustard are dominated by reactions with nucleophiles at its two electrophilic carbon atoms, plus oxidation of the electron-rich sulphur atom. Nucleophilic reactions proceed by an internal SN1 type mechanism, via the episulphonium ion shown in Figure 2.

The formation of the episulphonium ion is rate limiting and occurs very rapidly in polar solvents (Bartlett and Swain, 1949). In a competitive environment, the episulphonium ion reacts preferentially with ‘soft’ nucleophiles such as thiols, although it will react with a broad range of soft and hard nucleophiles. Under physiological conditions, reaction with the cysteinyl thiol function in glutathione (probably chemical rather than enzyme-mediated) competes with hydrolysis and reactions with nucleophilic sites on macromolecues. Metabolism studies in the rat with 35S- and 13C-labelled sulphur mustard showed that the initial reaction products with glutathione are metabolized by two divergent pathways to produce mercapturic acids (N-acetylcysteine conjugates) and β-lyase metabolites (methylthio/methylsulphinyl conju- gates) (Black et al., 1992a). The mustard sulphur atom in most metabolites is oxidized to sulphox- ide or sulphone. The various permutations of

Figure 3. Urinary metabolites of sulphur mustard that have been investigated as biomarkers

reactions with nucleophiles, on one or both electrophilic carbon atoms, combined with three pos- sible oxidation states of the sulphur atom, produce a large number of metabolites (at least 20 by HPLC using radioactivity detection). Figure 3 shows 5 of the 9 metabolites identified (1 — 5), which are the ones that have so far been investigated as biological markers. Not shown are three monomercapturic acid conjugates and mustard sulphoxide, which is much less reactive than mustard and was a very minor metabolite. These metabolites were identified by offline mass spectrometry following isolation by preparative HPLC, before the introduction of modern LC — MS systems that use atmospheric pressure ionization. Many more metabolites (e.g. derived from the partial hydrolysis product, hemi-mustard) could be identified using current instrumentation. Metabolism by the mercapturic acid pathway to the bis-N-acetylcysteine conjugate of mustard sulphone (3) is consistent with early studies by Roberts and Warwick (1963), in which TLC and dilution assays were used for tentative identification. The β-lyase metabo- lites 4 and 5 are formed through cleavage of the S — C bond in an intermediate bis-cysteinyl conjugate by the enzyme β-lyase, followed by

S-methylation (Bakke and Gustafsson, 1984). This metabolic pathway is believed to be mediated predominantly by gut flora. Of the metabo- lites derived from simple hydrolysis, thiodigly- col sulphoxide (TDGO) (2) was the major one, with much lower amounts of TDG (1). A separate metabolism study of TDG in the rat showed that > 90% is excreted as TDGO (Black et al., 1993). An early metabolism study using IV administration in the rat reported excretion of the bis-glutathione conjugate of sulphur mustard (Davison et al., 1961).

In a quantitative study of urinary excretion in rats following PC exposure to sulphur mustard, 3.7 — 13.6% of the dose was excreted as products of hydrolysis, mainly as TDGO, and 2.5 — 5.3% as β-lyase metabolites, but with considerable variation between animals (Black et al., 1992b). The excretion of β-lyase metabolites showed a sharper decline than that of hydrolysis products, suggesting that the prolonged excretion of the latter results from TDG being slowly liberated from adducts with macromolecules, e.g. from esters formed with aspartic and glutamic acid residues. A satisfactory analytical method for the bis-mercapturic acid conjugate (3) was not then available.

Figure 4. Additional metabolites of sulphur mustard

Two other urinary metabolites have been reported in animal studies (Figure 4). N7- (2-Hydroxyethylthioethyl)guanine (6), derived from the breakdown of alkylated DNA, was detected in rats (Fidder et al., 1996). The un- usual metabolite (7), assumed to result from re- action with a histidine residue, was identified in the pig following percutaneous administration (Sandelowsky et al., 1992).

Reactions with proteins

Haemoglobin

Albumin

(7)

sulphur mustard to give 2-hydroxyethylthioethyl esters (Capacio et al., 2004). Adducts with albu- min histidine residues have not been reported.

Keratin

Reaction with DNA

Analytical methods

Urinary Metabolites

Figure 5. Selective cleavage of alkylated N-terminal valine with pentafluorophenyl isothiocyanate

or GC — MS — MS after derivatization. The bispentafluorobenzoyl derivative in combination with NICI-MS (Black and Read, 1988,1995a), and the bis-heptafluorobutyryl derivative in com- bination with electron ionization or positive ion chemical ionization (Jakubowski et al., 1990; Boyer et al., 2004; Riches et al., 2007), provide the most sensitive methods, with limits of detection (LODs) down to ∼ 0.1 ng/ml. Conversion to the bistrimethylsilyl or tertbutyldimethylsilyl derivative is widely used in environmental analy- sis but these give higher LODs in urine (Ohsawa et al., 2004). TDGO is most easily analyzed after reduction to TDG with titanium trichloride (Black and Read, 1991). This is because of the difficulty of isolation of this very polar metabolite from aqueous media, and the different reactions involving the sulphoxide function that may occur on derivatization (Black and Muir, 2003). TDG and TDGO can be analyzed by LC — MS but detection limits have been too high for biomedical sample analysis. The β-lyase metabolites are readily analyzed by GC — MS and GC — MS — MS, provided that the sulphoxide groups, as with TDGO, are reduced with tita- nium trichloride (Black et al., 1991; Black and Read 1995a; Young et al., 2004). This produces the single analyte O2 S(CH2 CH2 SCH3 )2 , which is easily extracted and analyzed (LOD ∼ 0.1 ng/ml). Thus, TDG, TDGO and the two β-lyase metabolites can be analyzed as two analytes, TDG and O2 S(CH2 CH2 SCH3 )2 , from the same aliquot of urine treated with titanium trichloride (Black and Read, 1995a; Boyer et al., 2004). Some TDG and TDGO is excreted as glucuronides, as indicated by increased levels after treatment of urine with glucuronidase. LC — MS — MS, using positive electrospray ionization (ESI), also provides a sensitive method for the β-lyase metabolites, detecting them individually as the original metabolites (Read and Black, 2004a). No satisfactory GC — MS method has been developed for the bis N -acetylcysteine conjugate (3), probably because of thermal instability, but LC — MS — MS using negative electrospray ionization provides acceptable detection limits (Read and Black, 2004b).

Protein Adducts

Haemoglobin

Albumin

DNA Adducts

Human exposures

Urinary Metabolites

Protein and DNA Adducts

Table 1. Analyses of samples from human casualties of deliberate or accidental exposure to sulphur mustard.

results were corroborated by immunochemical analysis for the DNA adduct in lymphocytes from the same blood samples (Benschop et al., 1997). The histidine adduct was detected at slightly higher concentrations than the valine adduct in those samples analyzed for both (Black et al., 1997a). In samples collected up to 10 days after exposure, signal to noise ratios for β-lyase metabolites were generally greater than those obtained for adducts, but after a longer period adducts are the superior biomarkers.

Non-Metabolized Sulphured Mustard

Nitrogen Mustards

Biological fate

Metabolism

In vitro studies of HN-2 incubated with rat and rabbit liver homogenates indicated N -demethylation to be a significant pathway (up to 7% in 120 min), as judged by the generation of formaldehyde (Trams and Nadkarni, 1956). Loss of one of the CH2 CH2 X substituents also oc- curred, as indicated by the generation of acetalde- hyde. In aqueous media, nitrogen mustards are hydrolysed to the corresponding ethanolamines (9) (Figure 6) and, like TDG, these are excre- tion products in rodents following exposure. No metabolites derived from conjugation of nitro- gen mustards with glutathione have yet been reported.

Ethanolamines were determined quantitatively following PC administration of nitrogen mustards in the rat. Excretion in urine up to 48 h accounted for < 0.1% of the applied doses of HN-1andHN-2,andupto∼0.3%ofHN-3 (absorbed doses were not determined) (Lemire et al., 2004). The ethanolamines appeared to be excreted unconjugated, as treatment of the urine with β-glucuronidase had no effect.

Metabolism studies with ethanolamines have been undertaken in the context of their use as industrial chemicals. Following PC and IV administration of N-methyldiethanolamine in the rat, a major fraction of the absorbed dose was excreted as unidentified urinary metabolites, with some unchanged N-methyldiethanolamine (Leung et al., 1996). Triethanolamine was excreted predominantly unmetabolized in mice following both IV and percutaneous administration (Stott et al., 2000).

Protein Adducts

Figure 6. Nitrogen mustards and their hydrolysis products

with the anti-cancer nitrogen mustard drugs melphalan and cyclophosphamide (Noort et al., 2002b). HN-2 alkylation of histidine residues in haemoglobin has been indicated (Fung et al., 1975) but no definitive studies have been reported. Nornitrogen mustard, HN(CH2CH2Cl)2, reacts with N-terminal valines in haemoglobin in vitro (Thulin et al., 1996).

DNA Adducts

Analytical methods

Metabolites

Protein and DNA Adducts

LEWISITE Biological fate

Metabolism

Protein Adducts

Analytical methods

Figure 7. Hydrolysis of lewisite I to chlorovinylarsonous acid and its derivatization to 1,3-dithioarsenolines (10) with 1,2-dithiols

temperature. 1,2 Ethanedithiol (Jakubowski et al., 1993; Logan et al., 1999), 1,3 propanedithiol (Wooten et al., 2002) and 2,3 dimercaptopropan-1-ol (British Anti- Lewisite, BAL) (Fidder et al., 2000) have been used for biomedical sample analysis. Unlike most derivatizing reagents, which are reactive electrophiles, thiols can derivatize in situ in the biomedical fluid or aqueous extract. In the case of BAL as a derivatizing agent, the free hydroxyl is subsequently converted to its heptafluorobutyryl derivative. CVAA can be concentrated from urine by C18 SPE, either before or after derivatization, or by solid phase microextraction after derivatization. The latter provides a very sensitive method. LC — MS of CVAA gives ill-defined peaks unless it is oxidized to the pentavalent arsonic acid (Black and Muir, 2003).

Lewisite bound to haemoglobin cysteine residues is displaced by conversion to the BAL derivative shown in Figure 7 (Fidder et al., 2000). Exposure of guinea pigs to lewisite (0.25 mg/kg, subcutaneous) could be demonstrated by whole blood analysis (i.e. adducts + CVAA) up to at least 240 h.

There have been no reported human exposures to lewisite for which biomedical samples have been analyzed.

Nerve Agents

Biological fate

Distribution and Metabolism

The metabolism of nerve agents is dominated by hydrolysis. This occurs to a small extent by chemical reaction with water, but is predominantly mediated by

Figure 8. Metabolic pathways of nerve agents

phosphorylphosphatases. The main hydrolytic pathways that occur in the body are shown in Figure 8. Alternative hydrolytic pathways are possible for VX (through P — O and O — C cleavage) but these have not been reported in animal studies. For phosphonofluoridates (sarin, soman and GF) and V agents, the major metabolites are alkyl methylphosphonic acids (11). Further hydrolysis to methylphosphonic acid (MPA) may proceed slowly, as indicated by analyses of victims of the Tokyo subway attack (Nakajima et al., 1998). In rats, only traces of MPA were observed in urine (Shih et al., 1994).

Tabun hydrolyses by two pathways, through P — N and P — CN cleavage. It has not yet been es- tablished which of these predominates in animals or humans. Further hydrolysis produces ethyl phosphoric acid and eventually phosphate, both of which are ubiquitous from other sources.

Hydrolysis of VX produces 2-diisopropy- laminoethanethiol, HSCH2 CH2 N(i Pr)2 . A meta- bolite (12), derived from enzymatic S- methylation of this hydrolysis product, was identified in human plasma following an assassination with VX (Tsuchihashi et al. 1998). Experiments in rats confirmed the rapid metabolic formation of (12) from HSCH2 CH2 N(i Pr)2 (Tsuchihashi et al., 2000). This metabolite has not been reported in urine. By analogy with β-lyase metabolites of sulphur mustard, it might be excreted as a sulphoxide.

Protein Adducts

Acetyl and butyrylcholinesterase

AChE and BuChE, which have half-lives of 5 — 16 days, provide excellent biomarkers, but with one disadvantage. With certain nerve agents, particularly soman, a rapid secondary reaction oc- curs within the active site, in which the phosphyl moiety is dealkylated. This process, known as ‘ageing’, leads to loss of structural information on the inhibitor, for example with soman the pinacolyl group is lost (Figure 9). It also results in a negatively charged phosphyl moiety, which is resistant to reactivation by oximes and fluoride ion. Half-times quoted for ageing of human red blood cell AChE in vitro are: soman, 2 — 6 min;

Figure 9. Nerve agent adducts with BuChE and their digestion with pepsin

sarin, 3h, 5h; tabun, 13h, >14h; GF40h, 7.5 h; VX, 48 h (Dunn et al., 1997).

Adducts with albumin

Figure 10. Adducts of G agents with a tyrosine residue on albumin

then followed a steep rise in the concentration of the adduct. Adducts with all four agents have been detected in guinea pigs exposed to 0.5 or 2 × LD50 doses. The tyrosine adducts do not appear to age rapidly and can still be detected after therapeutic treatment with oximes (which should substantially reduce the amounts of non-aged phosphylated AChE and BuChE). Non-aged adducts with soman and tabun were detected 7 days after exposure to 5 × LD50 doses after treatment with oxime, atropine and anticonvulsant. Consistent with the reaction being entirely chemical, a tyrosine adduct with the less reactive VX was observed in blood in vitro only at high con-centrations. The probable formation of tyrosine adducts in a non-human primate is supported by the fluoride regeneration of soman (2.4 ng/ml) from plasma 3 days after an exposure to 2 × LD50 (intramuscular), when all of the inhibited BuChE should have aged (Adams et al., 2004). These studies also showed that agent could be regenerated from incubates of agent with human serum albumin. Albumin was recently shown to bind a biotinylated organophosphorus agent in mice, with diisopropyl phosphate and some organophosphorus pesticides competing for the same site (Peeples et al., 2005).

Analytical methods

Metabolites

Protein Adducts

Mass spectrometric detection of phosphylated BuChE provides a much more specific biomarker of exposure. BuChE is usually preferred to AChE because it is much more abundant in blood plasma than AChE is in erythrocytes. A versatile method of detection involves isolation of BuChE from plasma using affinity SPE, digestion with pepsin, and LC — MS — MS detection of a phosphylated nonapeptide encompassing the active site serine (Fidder et al., 2002). In the case of aged residues, this method will only identify part of the structure of the nerve agent although, provided it is a phosphonofluoridate or V agent, a methylphosphonyl MeP(O)-residue will be a clear indication that a nerve agent has been used (there are no pesticides in use that include a Me — P(O) moiety; fonofos has Et-P(O)).

Figure 11. Fluoride induced regeneration of sarin from sarininhibited BuChE in plasma of a rhesus monkey after IV administration. The rhesus monkey received a final dose of 0.7 μg/kg (taken from Van der Schans, M.J. et al., 2004). Reprinted from Archives of Toxicology, 78: 2004, 508–524, ‘Retrospective detection of exposure to nerve agents’, by M. J. Van der Schans et al., original copyright notice with kind permission of Springer Science and Business Media

An alternative, and experimentally less demanding method, displaces the organophosphorus moiety as a fluoridate (e.g. with sarininhibited BuChE the original nerve agent is regenerated), either from plasma BuChE (Polhuijs et al., 1997) or red cell AChE (Jakubowski et al., 2004). The alkyl methylphosphonyl fluoridate is readily extracted and detected using GC — MS (or GC — FPD). Fluoride reactivation is currently the most sensitive method for detecting ChE inhibition by nerve agents, enabling detection in blood samples with < 1 % BuChE inhibition (Degenhardt et al., 2004). The method has been demonstrated to be effective for GA, GB, GF and VX. Figure 11 shows detection up to 56 days following exposure of atropinized rhesus monkeys to a dose producing an initial 40% inhibition of BuChE (Van der Schans et al., 2004).

In the case of sarininhibited ChE, the phosphyl moiety has also been displaced as isopropyl methylphosphonic acid using trypsin digestion and alkaline phosphatase (Nagao et al., 1997; Matsuda et al., 1998).

Albumin adducts can be detected by LC-MS- MS as phosphylated tyrosine, after digestion of the albumin fraction with Pronase and SPE clean- up (Harrison et al., 2000b).

Human exposures

Metabolites

Table 2. Analysis of samples from casualties of Japanese terrorist incidents, exposed to sarin or VX
Figure 12. BZ and its hydrolysis products

of sarin in a comatose patient and 0.016 — 0.032 mg in less severely intoxicated patients. i PrMPA was detected by LC — MS — MS in serum collected within 2 h of hospitalization at concentrations of 3 — 136 ng/ml in 4 casualties of the Matsumoto incident and 2 — 100 ng/ml in 13 casualties of the Tokyo attack (Noort et al., 1998). High levels of iPrMPA correlated with low levels of BuChE activity. Ethyl methylphosphonic acid and 2-diisopropylaminoethyl methyl sulphide (12) were detected by GC — MS and GC — MS — MS in the serum of a subject assassinated by application of VX from a disposable syringe to the neck (Tsuchihashi et al., 1998, 2000).

Protein Adducts

QUINUCLIDINYL BENZILATE, BZ

Biological fate

Unlike the vesicants and nerve agents, BZ is not a reactive electrophile (other than with regard to hydrolysis) and no adducts are known to be formed either directly or indirectly through reactive metabolites.

Analytical methods

Phosgene

Biological fate

Metabolism

Protein Adducts

Analytical methods

Hydrogen Cyanide

Biological fate

Distribution and Metabolism

Figure 13. Metabolism of hydrogen cyanide

A small percentage reacts with vitamin B12, ap- pearing as cyanocobalamin in urine.

Analytical methods

Human exposures

Riot Control Agents

CS

Biological fate

Metabolism

The metabolism of CS is dominated by two pathways, the major one resulting from initial hydrolysis to 2-chlorobenzaldehyde and malononitrile (Figure 14), and the minor one from reduction to 2-chlorobenzylmalononitrile (dihydro- CS) (Figure 15) (Brewster et al., 1987). 2-Chlorobenzaldehyde (14) is further metabolized by hepatic oxidation to 2-chlorobenzoic acid (15), which is conjugated with glycine to form the major urinary metabolite 2-chlorohippuric acid (16). A minor metabolic pathway of 2-chlorobenzaldehyde is through reduction to 2-chlorobenzyl alcohol (17), which is excreted as glucuronide (18) and the N -acetylcysteine conjugate (19). The formation of the latter is postulated to occur via a sulphate intermediate (Rietveld et al., 1983). In these metabolic pathways, three of the carbon atoms of CS are lost as malononitrile, which is further metabolized to cyanide and thiocyanate. In the second metabolic pathway (Figure 14), CS is reduced to the hydrolytically more stable dihydro-CS (20), which is metabolized by hydrolysis with decarboxylation and excreted as the glycine conjugate (21), or by simple hydrolysis to the corresponding carboxamide

Figure 14. Metabolic pathway of CS initiated by hydrolysis

(22) and carboxylic acid (23). In this pathway, the carbon skeleton is either fully retained or loses two carbon atoms. No protein adducts of CS have been reported.

Human exposures

CN

Figure 15. Metabolic pathway of CS initiated by reduction

reacts faster with sulphur based nucleophiles than with water, but still at a slower rate than CS. The reaction products of CN with nucleophiles have been poorly characterized. Although it has been in use much longer than CS, no metabolism studies appear to have been reported. It will inhibit a number of sulphydryl based enzymes such as lactic dehydrogenase (Mackworth, 1948), and so it may form covalent adducts with blood proteins.

CR

Metabolism

Minor pathways arise from hydrolytic or oxidative cleavage of the azomethine moiety and reduction to dihydro-CR (French et al., 1983b). No samples from human exposures have been reported.

Capsaicin

Metabolism

Figure 16. Major metabolic pathway of CR, with excretion occurring mainly as sulphates in the rat and guinea pig and mainly as non-conjugated species in the rhesus monkey
Figure 17. Capsaicin and its urinary metabolite ω-hydroxycapsaicin

of rabbits following IV administration of capsaicin (Surh et al., 1995). Following oral administration of dihydrocapsaicin (20 mg/kg) in the rat, ∼ 75% of the dose was eliminated in the urine as unchanged dihydrocapsaicin plus eight metabolites (Kawada and Iwai, 1985). The metabolites were all derived from an initial hydrolytic cleavage of the carboxamide function and consisted of free metabolites (14.5% of the total dose) and glucuronide conjugates (60.5%). The following were identified: dihydrocapsaicin (8.7%), vanillamine (29) (4.7%), vanillin (30) (4.6%), vanillyl alcohol (31) (37.6%) and vanillic acid (32) (19.2%) (Figure 18). Dihydrocapsaicin hydrolyzing enzyme activity was found in various organs of the rat, but particularly in the liver and gut.

The other product of hydrolysis is initially 8- methylnonanoic acid, but it is not clear to what extent this is metabolized.

One of the concerns for capsaicins as food components is the potential for the aromatic phenolic moiety to be oxidised by P450 enzymes to potentially carcinogenic epoxide, phenoxy radical or quinone type electrophilic intermediates. Detailed studies of the metabolism of capsaicin in vitro, by recombinant cytochrome P450 enzyme and hepatic and lung microsomes, were reported by Reilly et al. (2003) and Reilly and Yost (2005). Metabolites were identified that were derived from aromatic and alkyl hydroxylation, aryl O-demethylation, alkyl dehydrogenation and an additional ring oxygenation. Addition of GSH to microsomal incubations with capsaicin trapped several reactive intermediates as GSH adducts, although these have not been reported as metabolites in animal studies.

Human exposures

Figure 18. Metabolites of dihydrocapsaicin identified in the rat (IG administration)

chilli spices. Vanillin, an intermediate in the hydrolytic pathway, is used extensively as a food additive.

In Conclusion

Detailed metabolism studies have been re- ported for the RCAs, CS and CR, and to a lesser extent, capsaicin, but sensitive analytical meth- ods for the metabolites have yet to be developed. The formation of covalent adducts with proteins has been little studied, although observations have suggested that CS and CN react with proteins. In the case of CS and capsaicin, major metabolites are derived from an initial hydrolysis with loss of some of the carbon skeleton, and it needs to be established if background levels of these metabolites occur in non-exposed individuals.

Analytical methods continue to be improved, and in some cases adapted for less costly instrumentation. At present, expertise in biomedical sample analysis for CW agents is restricted to a small number of laboratories. Recent interest shown by the OPCW, and the concern for terrorist use of CW, may encourage a larger number of laboratories to acquire expertise.

Business Chief Executive ( BCE ) at Flash Crypto Finance Government Protocol

Love podcasts or audiobooks? Learn on the go with our new app.