Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure.

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry.

Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 × 10-8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 µM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.

Verification of Exposure to Novichok Nerve Agents Utilizing a Semitargeted Human Butyrylcholinesterase Nonapeptide Assay.

Novichok (NV) nerve agents were recently added to the list of Schedule 1 chemicals of the Chemical Weapons Convention. There is a well-accepted method for assessment of nerve agent exposure based on mass spectrometric analysis of a nonapeptide with the serine-198 residue modified by the nerve agent, but this approach has not yet been reported for the class of NV agents and requires the availability of reference standards, which may be a limitation for NV agent exposure assessment. Thus, a goal of this study was to first verify the utility of the nonapeptide method for the characterization of human plasma samples exposed in vitro to the NV agents A-230, A-232, and A-234. A second aim was to evaluate the possibility of identifying unknown exposures by applying precursor ion scanning in combination with high resolution mass spectrometry (HRMS). Thus, precursor ion scanning, with a generic fragment ion (m/z 778) of the nonapeptide, was used to pinpoint any modified nonapeptide, while HRMS was used for structural elucidation of the adduct moiety. By this approach, use of HRMS enabled differentiation between adducts of agents with similar molecular masses. A new unique feature that could be exploited for NV nonapeptide analysis was that the modification was released from the peptide during fragmentation in the mass spectrometer and was detected in the low-mass region of the mass spectrum. This low-mass region was extremely informative and contributed to the assignment of the structure of the particular agent used, which is especially important in case no reference materials are available. The presented method is important for verification purposes by the Organisation for Prohibition of Chemical Weapons (OPCW), e.g., in case of investigations of alleged use of NV agents, and for regular forensic investigations.

Mass spectrometric analysis of adducts of sulfur mustard analogues to human plasma proteins: approach towards chemical provenancing in biomedical samples.

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure.

Oxidative stress leads to the activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. While most studies have focused on the activation of the Nrf2 pathway after single chemical treatment, little is known about the dynamic regulation of the Nrf2 pathway in the context of repeated exposure scenarios. Here we employed single cell live imaging to quantitatively monitor the dynamics of the Nrf2 pathway during repeated exposure, making advantage of two HepG2 fluorescent protein reporter cell lines, expressing GFP tagged Nrf2 or sulfiredoxin 1 (Srxn1), a direct downstream target of Nrf2. High throughput live confocal imaging was used to measure the temporal dynamics of these two components of the Nrf2 pathway after repeated exposure to an extensive concentration range of diethyl maleate (DEM) and tert-butylhydroquinone (tBHQ). Single treatment with DEM or tBHQ induced Nrf2 and Srxn1 over time in a concentration-dependent manner. The Nrf2 response to a second treatment was lower than the response to the first exposure with the same concentration, indicating that the response is adaptive. Moreover, a limited fraction of individual cells committed themselves into the Nrf2 response during the second treatment. Despite the suppression of the Nrf2 pathway, the second treatment resulted in a three-fold higher Srxn1-GFP response compared to the first treatment, with all cells participating in the response. While after the first treatment Srxn1-GFP response was linearly related to Nrf2-GFP nuclear translocation, such a linear relationship was less clear for the second exposure. siRNA-mediated knockdown demonstrated that the second response is dependent on the activity of Nrf2. Several other, clinically relevant, compounds (i.e., sulphorophane, nitrofurantoin and CDDO-Me) also enhanced the induction of Srxn1-GFP upon two consecutive repeated exposure. Together the data indicate that adaptation towards pro-oxidants lowers the Nrf2 activation capacity, but simultaneously primes cells for the enhancement of an antioxidant response which depends on factors other than just Nrf2. These data provide further insight in the overall dynamics of stress pathway activation after repeated exposure and underscore the complexity of responses that may govern repeated dose toxicity.

Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).

Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.

Verification of exposure to chemical warfare agents through analysis of persistent biomarkers in plants.

The continuing threats of military conflicts and terrorism may involve the misuse of chemical weapons. The present study aims to use environmental samples to find evidence of the release of such agents at an incident scene. A novel approach was developed for identifying protein adducts in plants. Basil (Ocimum basilicum), bay laurel leaf (Laurus nobilis) and stinging nettle (Urtica dioica) were exposed to 2.5 to 150 mg m-3 sulfur mustard, 2.5 to 250 mg m-3 sarin, and 0.5 to 25 g m-3 chlorine gas. The vapors of the selected chemicals were generated under controlled conditions in a dedicated set-up. After sample preparation and digestion, the samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS), respectively. In the case of chlorine exposure, it was found that 3-chloro- and 3,5-dichlorotyrosine adducts were formed. As a result of sarin exposure, the o-isopropyl methylphosphonic acid adduct to tyrosine could be analyzed, and after sulfur mustard exposure the N1- and N3-HETE-histidine adducts were identified. The lowest vapor exposure levels for which these plant adducts could be detected, were 2.5 mg m-3 for sarin, 50 mg m-3 for chlorine and 12.5 mg m-3 for sulfur mustard. Additionally, protein adducts following a liquid exposure of only 2 nmol Novichock A-234, 0.4 nmol sarin and 0.2 nmol sulfur mustard could still be observed. For both vapor and liquid exposure, the amount of adduct formed increased with the level of exposure. In all cases synthetic reference standards were used for unambiguous identification. The window of opportunity for investigation of agent exposure through the analysis of plant material was found to be remarkably long. Even three months after the actual exposure, the biomarkers could still be detected in the living plants, as well as in dried leaves. An important benefit of the current method is that a relatively simple and generic sample work-up procedure can be applied for all agents studied. In conclusion, the presented work clearly demonstrates the possibility of analyzing chemical warfare agent biomarkers in plants, which is useful for forensic reconstructions, including the investigation into alleged use in conflict areas.

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents.

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.

'Inverted' analogs of the antibiotic gramicidin S with an improved biological profile.

A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.

Exploring the conformational and biological versatility of ß-turn-modified gramicidin S by using sugar amino acid homologues that vary in ring size.

Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the ß-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.

Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives.

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.

Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

Gramicidin S derivatives containing cis- and trans-morpholine amino acids (MAAs) as turn mimetics.

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.

An adamantyl amino acid containing gramicidin S analogue with broad spectrum antibacterial activity and reduced hemolytic activity.

The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.

Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.

Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

Structural and biological evaluation of some loloatin C analogues.

Loloatin C is a cyclic cationic antimicrobial peptide which is active against gram positive as well as certain gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.

Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified D-phenylalanine residues.

The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified D-phenylalanine residue, their antibacterial properties against several gram positive and negative strains, as well as their hemolytic activity is reported.

Correction: Field, M. et al. AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients.

The authors wish to make the following corrections to their paper [...].