Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin.

A standard operating procedure has been developed for an immunoslotblot assay of sulfur mustard adducts to DNA in human blood and skin for use in a field laboratory. A minimum detectable level of exposure of human blood in vitro (> or = 50 nM) sulfur mustard is feasible with the assay. In the case of human skin, a 1 s exposure to saturated sulfur mustard vapor (830 mg/m(-3)) could still be detected.

Procedure for monitoring exposure to sulfur mustard based on modified edman degradation of globin.

A procedure for the modified Edman degradation of globin for determination of sulfur mustard adducts to the N-terminal valine residue in human hemoglobin has been developed for use under field laboratory conditions. The minimum detectable exposure level of human blood (in vitro) to sulfur mustard using this procedure is 100 nM. The interindividual and intraindividual variabilities of the procedure were acceptable (standard deviation < 10% and < 20%, respectively). The procedure could be properly set up and carried out in another laboratory within one working day, demonstrating its robustness.

Biochemical effects of low level exposure to soman vapour.

The aim of this study was to demonstrate changes in acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities, tyrosine aminotransferase activity (TAT) and plasma corticosterone level, neuroexcitability and behavior following 24 hours and 4 weeks of soman sublethal inhalation exposure at low level. AChE activity in erythrocytes and BuChE activity in plasma was decreased (dependent on the concentration of soman) 24 h and 4 weeks after the exposure. Similar decrease in AChE activity in different brain parts was observed. One of stressogenic parameters (TAT) was changed after 24 h exposure only. 4 weeks after the exposure, these parameters (corticosterone and TAT) were in the range of normal values. Behaviour of experimental animals was changed 24 h after the exposure persisting 4 weeks after the exposure as well as neuroexcitability.

In vitro adduct formation of phosgene with albumin and hemoglobin in human blood.

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [(14)C]phosgene (0-750 microM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide O=C-(V-L)-S-P-A, representing amino acid residues 1-5 of alpha-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiple-reaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of >/=1 microM phosgene.

Biomonitoring of exposure to lewisite based on adducts to haemoglobin.

The development of a procedure for retrospective detection and quantitation of exposure to the arsenical dichloro(2-chlorovinyl)arsine (lewisite; L1) has been initiated. Upon incubation of human blood with [14C]L1 (20 nM-0.2 mM) in vitro, more than 90% of the total radioactivity was found in the erythrocytes and 25-50% of the radioactivity becomes associated with globin. Evidence was obtained for the presence of several binding sites. One type of binding was identified as L1-induced crosslinking of cysteine residues 93 and 112 of the beta-globin chain. A method was developed for extraction of bound and unbound 2-chlorovinylarsonous acid (CVAA), a major metabolite of L1, from whole blood after treatment with 2,3-dimercapto-1-propanol (BAL). Subsequent to derivatization with heptafluorobutyryl imidazole, the CVAA-BAL derivative could be analysed at a 40-fmol level by means of gas chromatography-mass spectroscopy (GC-MS) under electron impact conditions. With this procedure, in vitro exposure of human blood to 1 nM L1 could be determined. The same procedure was applied to the analysis of human urine samples spiked with CVAA. In vivo exposure of guinea pigs could be established at least 240 h after subcutaneous administration of the agent (0.25 mg/kg) by the determination of bound and unbound CVAA in the blood. In the urine of these animals, CVAA could be detected for 12 h after exposure.

Diagnosis and dosimetry of exposure to sulfur mustard: development of a standard operating procedure for mass spectrometric analysis of haemoglobin adducts: exploratory research on albumin and keratin adducts.

Experiments were carried out to develop a standard operating procedure for analysis of sulfur mustard adducts to the N-terminal valine in haemoglobin and to explore adduct formation with albumin and keratin. In the first approach, gas chromatography-negative chemical ionization/mass spectrometry (GC-NCI/MS) of the thiohydantoin sample subsequent to the modified Edman degradation was performed using a thermodesorption/cold trap (TCT) injection technique (detection limit for in vitro exposure of human blood to sulfur mustard: 30 nM). In the second approach, the crude thiohydantoin sample was purified by solid-phase extraction procedures. In the third approach, the procedure was shortened significantly by performing the Edman degradation for 2 h at 60 degrees C. Upon exposure of human blood to various concentrations of [14C]sulfur mustard, ca. 20% was covalently bound to albumin. One of the tryptic fragments (T5 containing an alkylated cysteine (HETE-(A-L-V-L-I-A-F-A-Q-Y-L-Q-Q-C-P-F-E-D-H-V-K); MW 2536 Da) could be detected sensitively with liquid chromatography/tandem mass spectrometry analysis (detection limits: > or =15 pg absolute and 1 microM for in vitro exposure of human blood). Upon exposure of human callus (suspensions in 0.9% NaCl; 500 mg ml(-1)) to various concentrations of [14C]sulfur mustard we found 15-20% of the added radioactivity covalently bound to keratin. Upon incubation with base, 80% of the bound radioactivity was split off as [14C]thiodiglycol. This result opens the way for sensitive mass spectrometric detection of sulfur mustard exposure of skin by gas chromatography/mass spectrometry of (derivatized) thiodiglycol.

Alkylation of human serum albumin by sulfur mustard in vitro and in vivo: mass spectrometric analysis of a cysteine adduct as a sensitive biomarker of exposure.

To develop a mass spectrometric assay for the detection of sulfur mustard adducts with human serum albumin, the following steps were performed: quantitation of the binding of the agent to the protein by using [(14)C]sulfur mustard and analysis of acidic and tryptic digests of albumin from blood after exposure to sulfur mustard for identification of alkylation sites in the protein. The T5 fragment containing an alkylated cysteine could be detected in the tryptic digest with micro-LC/tandem MS analysis. Attempts to decrease the detection limit for in vitro exposure of human blood by analysis of the alkylated T5 fragment were not successful. After Pronase treatment of albumin, S-[2-[(hydroxyethyl)thio]ethyl]Cys-Pro-Phe was analyzed by means of micro-LC/tandem MS, allowing a detection limit for in vitro exposure of human blood of 10 nM, which is 1 order of magnitude lower than that obtained by means of modified Edman degradation. The analytical procedure could be successfully applied to the analysis of albumin samples from Iranian victims of the Iran-Iraq war.

Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers.

The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.

Low level nose-only exposure to the nerve agent soman: toxicokinetics of soman stereoisomers and cholinesterase inhibition in atropinized guinea pigs.

In order to initiate a quantitative basis for the toxicology of low level exposure to nerve agents, the toxicokinetics of soman stereoisomers during nose-only exposure for 5 h to 20 ppb (160 microg/m3) of C(+/-)P(+/-)-soman in air were studied in restrained, anesthetized, and atropinized guinea pigs. The concentrations of the toxic C(+/-)P(-)-soman stereoisomers in blood increased according to a biexponential function, after an initial lag time of ca. 30 min for C(+)P(-)-soman, with final concentrations

Verification of exposure to sulfur mustard in two casualties of the Iran-Iraq conflict.

The exposure of two Iranian victims of the Iran-Iraq conflict (1980-1988) to sulfur mustard was established by immunochemical and mass spectrometric analysis of blood samples taken 22 and 26 days after alleged exposure. One victim suffered from skin injuries compatible with sulfur mustard intoxication but did not have lung injuries; the symptoms of the other victim were only vaguely compatible with sulfur mustard intoxication. Both patients recovered. Immunochemical analysis was based on detection of the N7-guanine adduct of the agent in DNA from lymphocytes and granulocytes, whereas the N-terminal valine adduct in globin was determined by gas chromatography-mass spectrometry after a modified Edman degradation. The valine adduct levels correspond with those found in human blood after in vitro treatment with 0.9 microM sulfur mustard.

Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray-ionization mass spectrometry.

While non-reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methylphosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64,700 Da (calculated 64,695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64,820, 64,840, 64,852 and 64,860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64,780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.

Development of a physiologically based model for the toxicokinetics of C(+/-)P(+/-)-soman in the atropinized guinea pig.

A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(+/-)P(+/-)-soman at doses corresponding to 0.8-6 LD50 in the atropinized guinea pig. The model differentiates between the summated highly toxic C(+/-)P(-)-soman stereoisomers at supralethal doses and the individual nontoxic C(+/-)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(+/-)P(-)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD50. In view of large differences in the rates of hydrolysis of the C(+/-)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(+/-)P(-)-soman at a dose of 0.8 LD50, the almost instantaneous elimination of the C(+)P(-)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(+/-)P(-)- and C(+/-)P(+)-isomers.

Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood.

As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.

Protection of guinea pigs against soman poisoning by pretreatment with p-nitrophenyl phosphoramidates.

Several p-nitrophenyl phosphoramidates with the general formula RO(NH2)P(O)OC6H4-p-NO2, in which R = CH2CH3, CH2CH2F, CH2CHF2, CH2CF3, CH2CH2CH2CH3, and CH2CH2Cl, as well as (NH2)2P(O)OC6H4-p-NO2 were administered intravenously to guinea pigs as pretreatment compounds for protection against the lethal effects of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) poisoning. Administration of phosphoramidates at a dose that produces 30% acetylcholinesterase (AChE) inhibition in the blood of atropinized guinea pigs at the moment of soman poisoning increases the subcutaneous LD50 of soman up to almost fivefold depending on which compound was used. A synergistic effect with atropine was observed. Three of these compounds offered a higher degree of protection against soman poisoning than pyridostigmine. Furthermore, the surviving animals pretreated with the two phosphoramidates that provided the highest protective ratio were in a better condition at 24 hr after soman intoxication than those pretreated with pyridostigmine. Due to the limited number of compounds and their different characteristics, it appeared difficult to demonstrate a relationship between the rate of spontaneous reactivation of AChE inhibited by the pretreatment compound and the protection against soman. Nevertheless, the results indicate that a several-fold decrease in the rate of spontaneous reactivation relative to that of pyridostigmine may increase the protective ratio against soman.

Characterization of sulfur mustard induced structural modifications in human hemoglobin by liquid chromatography--tandem mass spectrometry.

In this paper we describe the use of tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to bis(2-chloroethyl) sulfide (sulfur mustard). Globin isolated from human whole blood which had been exposed to sulfur mustard was degraded with trypsin, and the digests were analyzed by micro LC/MS. Alkylated tryptic fragments (alpha-T1, alpha-T4, alpha-T6, alpha-T9, beta-T1, beta-T9, beta-T10, beta-T11, and beta-T10-S-S-beta-T12) could be tentatively assigned upon comparison with a digest from nonexposed globin. Subsequent tandem mass spectrometry of these peptides allowed unambiguous assignment of 5 specific modified residues: alpha-Val-1, alpha-His-20, beta-Val-1, beta-His-77, and beta-His-97. The results demonstrate the usefulness of microbore LC in combination with tandem mass spectrometry for the structural determination of chemically modified peptides and proteins.

Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation.

We report that exposure to the chemical warfare agent sulfur mustard can be monitored by means of a modified Edman degradation involving selective release of the N-terminal valine adduct of hemoglobin with the agent. The degree of alkylation of the N-terminal valine in human hemoglobin is approximately 1-2% of the total alkylation induced in hemoglobin upon treatment of human blood with sulfur mustard. After modified Edman degradation, followed by derivatization with heptafluorobutyric anhydride, the obtained pentafluorophenyl thiohydantion derivative of the valine adduct could be analyzed at a > or = 0.5 fmol level by means of GC/MS under negative ion chemical ionization conditions. Applying this procedure, in vitro exposure of human blood to > or = 0.1 microM of sulfur mustard could be determined. In vivo exposure of guinea pigs could also be established at 48 h after intoxication intravenously with 0.5 mg/kg (0.06 LD50) of the agent.

Solid-phase synthesis of peptide haptens containing a cysteine-sulfur mustard adduct.

Solid-phase synthesis of peptide haptens containing 2-[2-(S-cysteinyl)ethanol has been achieved by solution-phase synthesis of a properly protected S-alkylated cysteine derivative and subsequent solid-phase incorporation.

Synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA of calf thymus and human blood.

As part of a program to develop methods for verification of alleged exposure to sulfur mustard, we synthesized and characterized the adducts most likely formed by alkylation of DNA with sulfur mustard: N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine (1), bis[2-(guanin-7-yl)ethyl] sulfide (2), N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine (3), and O6-[2-[(2-hydroxyethyl)thio]ethyl]-guanine and its 2'-deoxyguanosine derivative. Incubation of double-stranded calf thymus DNA and human blood with [35S]sulfur mustard in vitro followed by enzymatic degradation of the DNA and mild depurination afforded three major radioactive peaks upon HPLC analysis. These peaks were identified as 1-3 by coelution with the synthetic markers and mass spectrometric and electronic spectra. Compound 1 appeared to be the most abundant adduct, which is in agreement with previous investigations on DNA alkylation with sulfur mustard.

Hydrolysis and binding of a toxic stereoisomer of soman in plasma and tissue homogenates from rat, guinea pig and marmoset, and in human plasma.

The fallen concentration of one of the two isomers of soman (1,2,2-trimethylpropyl methylphosphonofluoridate), i.e., C(+)P(-)-soman, was investigated in plasma and in homogenates of brain, lung, liver, kidney, diaphragm, skeletal muscle and mucosa of small intestines from rat, guinea pig and marmoset, and in human plasma (pH 7.5, 37 degrees). The decrease of the isomer concentration was followed by gas chromatographic determination of the residual concentration and proceeded in two phases due to a very rapid saturation of covalent binding sites for the isomer followed by catalysed hydrolysis. Estimates for the concentrations of covalent binding sites were obtained, which were relatively high in liver and kidney. Time periods for the hydrolysis of the isomer from a concentration of 40 ng/mL to 20 ng/mL were evaluated from the second reaction phase. It is concluded that the spontaneous and enzyme-catalyzed hydrolytic activities found for degradation of C(+)P(-)-soman in organs participating in central elimination are sufficiently high to account for the terminal half-life times of the isomer found in our toxicokinetic studies for the blood concentration after intoxication with 2-6 LD50 C(+/-)P(+/-)-soman. The hydrolytic activities are lower in the target organs for toxic action of soman, e.g., diaphragm and brain, especially for guinea pigs and marmosets.