A nested case-control study of untargeted albumin adductomics and acute myeloid leukemia.

Environmental exposures often produce reactive electrophiles in vivo, leading to oxidative stress, which plays a major role in carcinogenesis. These electrophiles frequently form adducts with human albumin, which can be measured to assess in vivo oxidative stress. Here, we aimed to examine the associations between circulatory albumin adducts and acute myeloid leukemia (AML), the most common adult myeloid leukemia that showed consistent associations with environmental exposures. We conducted a nested case-control study of 52 incident AML cases and 103 controls matched on age, sex and race within two prospective cohorts: the CLUE and PLCO studies. We measured 42 untargeted albumin adducts in prediagnostic samples using liquid chromatography-high-resolution mass spectrometry. Circulatory albumin adducts were associated with AML in conditional logistic regression models. For instance, higher levels of Cys34 disulfide adduct of the S-γ-glutamylcysteine, a precursor of the essential antioxidant, glutathione were associated with a lower risk of AML (odds ratios [95% confidence intervals]) for the 1st, 2nd and 3rd tertiles were 1.0, 0.65 (0.31-1.36) and 0.31 (0.12-0.80), respectively (P-trend = .01). These associations were largely driven by effects present among cases diagnosed at or above the median follow-up time of 5.5 years. In conclusion, applying a novel approach to characterize exposures in the prediagnostic samples, we found evidence supporting the notion that oxidative stress may play a role in the pathogenesis of AML. Our findings offer insight into AML etiology and may be relevant in identifying novel therapeutic targets.

Extending the HSA-Cys34-Adductomics Pipeline to Modifications at Lys525.

We previously developed an adductomics pipeline that employed nanoflow liquid chromatography and high-resolution tandem mass spectrometry (nLC-HR-MS/MS) plus informatics to perform an untargeted detection of modifications to Cys34 in the tryptic T3 peptide of human serum albumin (HSA) (21ALVLIAFAQYLQQC34PFEDHVK41). In order to detect these peptide modifications without targeting specific masses, the pipeline interrogates MS2 ions that are signatures of the T3 peptide. The pipeline had been pilot-tested with archived plasma from healthy human subjects, and several of the 43 Cys34 adducts were highly associated with the smoking status. In the current investigation, we adapted the pipeline to include modifications to the ε-amino group of Lys525─a major glycation site in HSA─and thereby extend the coverage to products of Schiff bases that cannot be produced at Cys34. Because trypsin is generally unable to digest proteins at modified lysines, our pipeline detects miscleaved tryptic peptides with the sequence 525KQTALVELVK534. Adducts of both Lys525 and Cys34 are measured in a single nLC-HR-MS/MS run by increasing the mass range of precursor ions in MS1 scans and including both triply and doubly charged precursor ions for collision-induced dissociation fragmentation. For proof of principle, we applied the Cys34/Lys525 pipeline to archived plasma specimens from a subset of the same volunteer subjects used in the original investigation. Twelve modified Lys525 peptides were detected, including products of glycation (fructosyl-lysine plus advanced-glycated-end products), acetylation, and elimination of ammonia and water. Surprisingly, the carbamylated and glycated adducts were present at significantly lower levels in smoking subjects. By including a larger class of in vivo nucleophilic substitution reactions, the Cys34/Lys525 adductomics pipeline expands exposomic investigations of unknown human exposure to reactive electrophiles derived from both exogenous and endogenous sources.

Agnostic Cys34-albumin adductomics and DNA methylation: Implication of N-acetylcysteine in lung carcinogenesis years before diagnosis.

Although smoking and oxidative stress are known contributors to lung carcinogenesis, their mechanisms of action remain poorly understood. To shed light into these mechanisms, we applied a novel approach using Cys34-adductomics in a lung cancer nested case-control study (n = 212). Adductomics profiles were integrated with DNA-methylation data at established smoking-related CpG sites measured in the same individuals. Our analysis identified 42 Cys34-albumin adducts, of which 2 were significantly differentially abundant in cases and controls: adduct of N-acetylcysteine (NAC, p = 4.15 × 10-3 ) and of cysteinyl-glycine (p = 7.89 × 10-3 ). Blood levels of the former were found associated to the methylation levels at 11 smoking-related CpG sites. We detect, for the first time in prospective blood samples, and irrespective of time to diagnosis, decreased levels of NAC adduct in lung cancer cases. Altogether, our results highlight the potential role of these adducts in the oxidative stress response contributing to lung carcinogenesis years before diagnosis.

Untargeted adductomics of Cys34 modifications to human serum albumin in newborn dried blood spots.

Metabolism of chemicals from the diet, exposures to xenobiotics, the microbiome, and lifestyle factors (e.g., smoking, alcohol intake) produce electrophiles that react with nucleophilic sites in circulating proteins, notably Cys34 of human serum albumin (HSA). To discover potential risk factors resulting from in utero exposures, we are investigating HSA-Cys34 adducts in archived newborn dried blood spots (DBS) that reflect systemic exposures during the last month of gestation. The workflow includes extraction of proteins from DBS, measurement of hemoglobin (Hb) to normalize for blood volume, addition of methanol to enrich HSA by precipitation of Hb and other interfering proteins, digestion with trypsin, and detection of HSA-Cys34 adducts via nanoflow liquid chromatography-high-resolution mass spectrometry. As proof-of-principle, we applied the method to 49 archived DBS collected from newborns whose mothers either actively smoked during pregnancy or were nonsmokers. Twenty-six HSA-Cys34 adducts were detected, including Cys34 oxidation products, mixed disulfides with low molecular weight thiols (e.g., cysteine, homocysteine, glutathione, cysteinylglycine), and other modifications. Data were normalized with a novel method ("scone") to remove unwanted technical variation arising from HSA digestion, blood volume, DBS age, mass spectrometry analysis, and batch effects. Using an ensemble of linear and nonlinear models, the Cys34 adduct of cyanide was found to consistently discriminate between newborns of smoking and nonsmoking mothers with a mean fold change (smoking/nonsmoking) of 1.31. These results indicate that DBS adductomics is suitable for investigating in utero exposures to reactive chemicals and metabolites that may influence disease risks later in life.

Adductomic signatures of benzene exposure provide insights into cancer induction.

Although benzene has long been recognized as a cause of human leukemia, the mechanism by which this simple molecule causes cancer has been problematic. A complicating factor is benzene metabolism, which produces many reactive intermediates, some specific to benzene and others derived from redox processes. Using archived serum from 20 nonsmoking Chinese workers, 10 with and 10 without occupational exposure to benzene (exposed: 3.2-88.9 ppm, controls: 0.002-0.020 ppm), we employed an adductomic pipeline to characterize protein modifications at Cys34 of human serum albumin, a nucleophilic hotspot in extracellular fluids. Of the 47 measured human serum albumin modifications, 39 were present at higher concentrations in benzene-exposed workers than in controls and many of the exposed-control differences were statistically significant. Correlation analysis identified three prominent clusters of adducts, namely putative modifications by benzene oxide and a benzene diolepoxide that grouped with other measures of benzene exposure, adducts of reactive oxygen and carbonyl species, and Cys34 disulfides of small thiols that are formed following oxidation of Cys34. Benzene diolepoxides are potent mutagens and carcinogens that have received little attention as potential causes of human leukemia. Reactive oxygen and carbonyl species-generated by redox processes involving polyphenolic benzene metabolites and by Cyp2E1 regulation following benzene exposure-can modify DNA and proteins in ways that contribute to cancer. The fact that these diverse human serum albumin modifications differed between benzene-exposed and control workers suggests that benzene can increase leukemia risks via multiple pathways involving a constellation of reactive molecules.

Untargeted lipidomic features associated with colorectal cancer in a prospective cohort.

BACKGROUND: Epidemiologists are beginning to employ metabolomics and lipidomics with archived blood from incident cases and controls to discover causes of cancer. Although several such studies have focused on colorectal cancer (CRC), they all followed targeted or semi-targeted designs that limited their ability to find discriminating molecules and pathways related to the causes of CRC. METHODS: Using an untargeted design, we measured lipophilic metabolites in prediagnostic serum from 66 CRC patients and 66 matched controls from the European Prospective Investigation into Cancer and Nutrition (Turin, Italy). Samples were analyzed by liquid chromatography-high-resolution mass spectrometry (LC-MS), resulting in 8690 features for statistical analysis. RESULTS: Rather than the usual multiple-hypothesis-testing approach, we based variable selection on an ensemble of regression methods, which found nine features to be associated with case-control status. We then regressed each selected feature on time-to-diagnosis to determine whether the feature was likely to be either a potentially causal biomarker or a reactive product of disease progression (reverse causality). CONCLUSIONS: Of the nine selected LC-MS features, four appear to be involved in CRC etiology and merit further investigation in prospective studies of CRC. Four other features appear to be related to progression of the disease (reverse causality), and may represent biomarkers of value for early detection of CRC.

Cys34 Adductomes Differ between Patients with Chronic Lung or Heart Disease and Healthy Controls in Central London.

Oxidative stress generates reactive species that modify proteins, deplete antioxidant defenses, and contribute to chronic obstructive pulmonary disease (COPD) and ischemic heart disease (IHD). To determine whether protein modifications differ between COPD or IHD patients and healthy subjects, we performed untargeted analysis of adducts at the Cys34 locus of human serum albumin (HSA). Biospecimens were obtained from nonsmoking participants from London, U.K., including healthy subjects (n = 20) and patients with COPD (n = 20) or IHD (n = 10). Serum samples were digested with trypsin and analyzed by liquid chromatography-high resolution mass spectrometry. Effects of air pollution on adduct levels were also investigated based on estimated residential exposures to PM2.5, O3 and NO2. For the 39 adducts with sufficient data, levels were essentially identical in blood samples collected from the same subjects on two consecutive days, consistent with the 28 day residence time of HSA. Multivariate linear regression revealed 21 significant associations, mainly with the underlying diseases but also with air-pollution exposures (p-value < 0.05). Interestingly, most of the associations indicated that adduct levels decreased with the presence of disease or increased pollutant concentrations. Negative associations of COPD and IHD with the Cys34 disulfide of glutathione and two Cys34 sulfoxidations, were consistent with previous results from smoking and nonsmoking volunteers and nonsmoking women exposed to indoor combustion of coal and wood.

Profiling the Serum Albumin Cys34 Adductome of Solid Fuel Users in Xuanwei and Fuyuan, China.

Xuanwei and Fuyuan counties in China have the highest lung cancer rates in the world due to household air pollution from combustion of smoky coal for cooking and heating. To discover potential biomarkers of indoor combustion products, we profiled adducts at the Cys34 locus of human serum albumin (HSA) in 29 nonsmoking Xuanwei and Fuyuan females who used smoky coal, smokeless coal, or wood and 10 local controls who used electricity or gas fuel. Our untargeted "adductomics" method detected 50 tryptic peptides of HSA, containing Cys34 and prominent post-translational modifications. Putative adducts included Cys34 oxidation products, mixed disulfides, rearrangements, and truncations. The most significant differences in adduct levels across fuel types were observed for S-glutathione (S-GSH) and S-γ-glutamylcysteine (S-γ-GluCys), both of which were present at lower levels in subjects exposed to combustion products than in controls. After adjustment for age and personal measurements of airborne benzo(a)pyrene, the largest reductions in levels of S-GSH and S-γ-GluCys relative to controls were observed for users of smoky coal, compared to users of smokeless coal and wood. These results point to possible depletion of GSH, an essential antioxidant, and its precursor γ-GluCys in nonsmoking females exposed to indoor-combustion products in Xuanwei and Fuyuan, China.

Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin.

An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short-lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating, and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a "housekeeping peptide", and annotates modifications based on a combination of retention time, accurate mass, elemental composition, and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race, and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g., ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.

Antibody enrichment and mass spectrometry of albumin-Cys34 adducts.

Untargeted analyses of tryptic peptides of human serum albumin (HSA) have been used to investigate unknown exposures to reactive electrophiles (adductomics). To reduce the complexity of the analytical matrix and thereby enhance identification of adducts by liquid chromatography-high-resolution mass spectrometry (LC-HRMS), a polyclonal anti-T3 antibody was designed to capture Cys34 adducts in tryptic digests of HSA (T3 is the third largest tryptic peptide). Epitopes were selected from sequences at both C- and N-termini based on the three-dimensional structure of the T3 peptide to minimize the influence of modified Cys34 residues. The assay was simplified by attaching magnetic beads to the anti-T3 antibody. When applied to commercial HSA and to plasma samples from healthy humans and analyzed by LC-HRMS, antibody treatment greatly reduced the background of non-T3 peptides in the sample matrix. Although other lipophilic HSA peptides were still present, presumably due to nonspecific binding to the antibody-magnetic-bead surfaces, their concentrations in antibody-treated samples were reduced about 6-fold compared to the same samples that had not been treated with the antibody. Analysis of antibody-enriched HSA digests from human plasma samples revealed 10 modified T3 peptides of which 8 were identified from accurate masses. Identified peptides included Cys34 oxidation and cysteinylation products and modifications representing losses of water and Lys and transpeptidation of Arg.

Placebo-resistant gut bacteria: Akkermansia muciniphila spp. and Familial Mediterranean fever disease.

Introduction: Despite numerous investigations into the impact of drugs/probiotics on the gut microbiota composition in Familial Mediterranean Fever (FMF) patients, the question as to whether there exists a significant bacterial diversity(ies) independent of the placebo effect that can be reliably considered in clinical and nutritional trials remains unresolved. Methods: This study represents the in augural analysis of the placebo's influence on the gut microbiota of both healthy individuals and FMF afflicted men, utilizing previously collected data from PhyloChip™ DNA microarray experiments. A total of 15 healthy and 15 FMF male volunteers, aged 18 to 50, participated in this partially randomized placebo trial, which is accessible through the GEO Series accession number GSE111835. Results and Discussion: Key findings from current investigations include i. the anticipated divergence in gut bacteria resistance to placebo between healthy and FMF individuals, ii. the minor impact of placebo on gut bacterial diversities in healthy individuals, with Enterobacteriaceae diversities identified as placebo-resistant among "healthy" gut bacteria, and iii. the comprehensive influence of placebo on all bacterial phyla in the gut microbiome of FMF patients, extending to nearly all bacterial genera, except for the resilience of gut Akkermansia muciniphila spp. to placebo in FMF patients. This study underscores the susceptibility of Faecalibacterium, Blautia, and Clostridium genera to placebo. Consequently, this investigation holds significance for the proper design of placebo-controlled trials and establishes a foundation for further exploration of the gut-brain axis. Furthermore, it contributes valuable insights to discussions regarding proposals for probiotic therapies, particularly focusing on Faecalibacterium spp., Blautia spp., and Clostridium spp.

Profiling Cys34 adducts of human serum albumin by fixed-step selected reaction monitoring.

A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins.

HSA Adductomics Reveals Sex Differences in NHL Incidence and Possible Involvement of Microbial Translocation.

BACKGROUND: The higher incidence of non-Hodgkin lymphoma (NHL) in males is not well understood. Although reactive oxygen species (ROS) have been implicated as causes of NHL, they cannot be measured directly in archived blood. METHODS: We performed untargeted adductomics of stable ROS adducts in human serum albumin (HSA) from 67 incident NHL cases and 82 matched controls from the European Prospective Investigation into Cancer and Nutrition-Italy cohort. Regression and classification methods were employed to select features associated with NHL in all subjects and in males and females separately. RESULTS: Sixty seven HSA-adduct features were quantified by liquid chromatography-high-resolution mass spectrometry at Cys34 (n = 55) and Lys525 (n = 12). Three features were selected for association with NHL in all subjects, while seven were selected for males and five for females with minimal overlap. Two selected features were more abundant in cases and seven in controls, suggesting that altered homeostasis of ROS may affect NHL incidence. Heat maps revealed differential clustering of features between sexes, suggesting differences in operative pathways. CONCLUSIONS: Adduct clusters dominated by Cys34 oxidation products and disulfides further implicate ROS and redox biology in the etiology of NHL. Sex differences in dietary and alcohol consumption also help to explain the limited overlap of feature selection between sexes. Intriguingly, a disulfide of methanethiol from enteric microbial metabolism was more abundant in male cases, thereby implicating microbial translocation as a potential contributor to NHL in males. IMPACT: Only two of the ROS adducts associated with NHL overlapped between sexes and one adduct implicates microbial translocation as a risk factor.

Cys34 adducts of reactive oxygen species in human serum albumin.

Long-term exposures to reactive oxygen species (ROS) have been linked to scores of chronic diseases. This has motivated interest in oxidation products of the only free cysteine residue (Cys34) of human serum albumin (HSA) as possible biomarkers of ROS exposure. However, Cys34 oxidation products have not been detected in human serum or plasma. Using liquid chromatography-high resolution tandem mass spectrometry, we report accurate masses and molecular compositions of Cys34 oxidation products in the 2432 Da peptide resulting from tryptic digestion of HSA. Peptides containing the expected sulfinic (Cys-SO(2)H) and sulfonic (Cys-SO(3)H) acids, as well as an adduct representing addition of one oxygen atom and loss of two hydrogen atoms, were detected in four archived samples of human plasma and one fresh sample of human serum. We speculate that this latter adduct is a sulfinamide formed by intramolecular reaction between either the Cys34 sulfenic acid (Cys-SOH) or sulfinic acid (Cys-SO(2)H) and the adjacent glutamine residue (Gln33). All three Cys34 adducts were measured in the five human samples with levels decreasing in the order sulfinic acid > (proposed) sulfinamide > sulfonic acid. Parallel measurements of a negative control detected only small amounts of the Cys34 sulfonic acid and the (proposed) sulfinamide and did not detect the sulfinic acid.

Exposure to diesel engine exhaust and alterations to the Cys34/Lys525 adductome of human serum albumin.

We investigated whether exposure to carcinogenic diesel engine exhaust (DEE) was associated with altered adduct levels in human serum albumin (HSA) residues. Nano-liquid chromatography-high resolution mass spectrometry (nLC-HRMS) was used to measure adducts of Cys34 and Lys525 residues in plasma samples from 54 diesel engine factory workers and 55 unexposed controls. An untargeted adductomics and bioinformatics pipeline was used to find signatures of Cys34/Lys525 adductome modifications. To identify adducts that were altered between DEE-exposed and unexposed participants, we used an ensemble feature selection approach that ranks and combines findings from linear regression and penalized logistic regression, then aggregates the important findings with those determined by random forest. We detected 40 Cys34 and 9 Lys525 adducts. Among these findings, we found evidence that 6 Cys34 adducts were altered between DEE-exposed and unexposed participants (i.e., 841.75, 851.76, 856.10, 860.77, 870.43, and 913.45). These adducts were biologically related to antioxidant activity.

Untargeted adductomics of newborn dried blood spots identifies modifications to human serum albumin associated with childhood leukemia.

The developing fetus is exposed to chemicals, which are metabolized to electrophiles that form adducts with nucleophilic Cys34 of human serum albumin (HSA). By measuring these adducts in neonatal blood spots (NBS), we obtain information regarding fetal exposures during the last month of gestation. To discover potential risk factors for childhood leukemia resulting from in utero exposures, we used untargeted adductomics to measure HSA-Cys34 adducts in 782 archived NBS, collected from incident cases of childhood acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) and matched population-based controls. Among a total of 28 Cys34 modifications that were measured, we found no differences in adduct abundances between childhood leukemia cases and controls overall. However, cases of T-cell ALL had higher abundances of adducts of reactive carbonyl species and a Cys34 disulfide of homocysteine was present at lower levels in AML cases. These results suggest that oxidative stress and lipid peroxidation may be etiologic factors of T-cell ALL, and alterations in one-carbon metabolism and epigenetic changes may be predictors of AML. Future replication of the results with larger sample sizes is necessary.

Relationships between airborne pollutants, serum albumin adducts and short-term health outcomes in an experimental crossover study.

Exposure to air pollution can have both short-term and long-term effects on health. However, the relationships between specific pollutants and their effects can be obscured by characteristics of both the pollution and the exposed population. One way of elucidating the relationships is to link exposures and internal changes at the level of the individual. To this end, we combined personal exposure monitoring (59 individuals, Oxford Street II crossover study) with mass-spectrometry-based analyses of putative serum albumin adducts (fixed-step selected reaction monitoring). We attempted to infer adducts' identities using data from another, higher-resolution mass spectrometry method, and were able to detect a semi-synthetic standard with both methods. A generalised least squares regression method was used to test for associations between amounts of adducts and pollution measures (ambient concentrations of nitrogen dioxide and particulate matter), and between amounts of adducts and short-term health outcomes (measures of lung health and arterial stiffness). Amounts of some putative adducts (e.g., one with a positive mass shift of ∼143 Da) were associated with exposure to pollution (11 associations), and amounts of other adducts were associated with health outcomes (eight associations). Adducts did not appear to provide a link between exposures and short-term health outcomes.

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase.

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.

Nanoimages show disruption of tubulin polymerization by chlorpyrifos oxon: implications for neurotoxicity.

Organophosphorus agents cause cognitive deficits and depression in some people. We hypothesize that the mechanism by which organophosphorus agents cause these disorders is by modification of proteins in the brain. One such protein could be tubulin. Tubulin polymerizes to make the microtubules that transport cell components to nerve axons. The goal of the present work was to measure the effect of the organophosphorus agent chlorpyrifos oxon on tubulin polymerization. An additional goal was to identify the amino acids covalently modified by chlorpyrifos oxon in microtubule polymers and to compare them to the amino acids modified in unpolymerized tubulin dimers. Purified bovine tubulin (0.1 mM) was treated with 0.005-0.1 mM chlorpyrifos oxon for 30 min at room temperature and then polymerized by addition of 1 mM GTP to generate microtubules. Microtubules were visualized by atomic force microscopy. Chlorpyrifos oxon-modified residues were identified by tandem ion trap electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry of tryptic peptides. Nanoimaging showed that low concentrations (0.005 and 0.01 mM) of chlorpyrifos oxon yielded short, thin microtubules. A concentration of 0.025 mM stimulated polymerization, while high concentrations (0.05 and 0.1 mM) caused aggregation. Of the 17 tyrosines covalently modified by chlorpyrifos oxon in unpolymerized tubulin dimers, only 2 tyrosines were labeled in polymerized microtubules. The two labeled tyrosines in polymerized tubulin were Tyr 103 in EDAANNYR of alpha tubulin, and Tyr 281 in GSQQYR of beta tubulin. In conclusion, chlorpyrifos oxon binding to tubulin disrupts tubulin polymerization. These results may lead to an understanding of the neurotoxicity of organophosphorus agents.

Mass spectrometry identifies multiple organophosphorylated sites on tubulin.

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.