Shank3 mutation in a mouse model of autism leads to changes in the S-nitroso-proteome and affects key proteins involved in vesicle release and synaptic function.

Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO•)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO• Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO•-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD.

Low Doses of Arsenic in a Mouse Model of Human Exposure and in Neuronal Culture Lead to S-Nitrosylation of Synaptic Proteins and Apoptosis via Nitric Oxide.

BACKGROUND: Accumulating public health and epidemiological literature support the hypothesis that arsenic in drinking water or food affects the brain adversely. METHODS: Experiments on the consequences of nitric oxide (NO) formation in neuronal cell culture and mouse brain were conducted to probe the mechanistic pathways of nitrosative damage following arsenic exposure. RESULTS: After exposure of mouse embryonic neuronal cells to low doses of sodium arsenite (SA), we found that Ca2+ was released leading to the formation of large amounts of NO and apoptosis. Inhibition of NO synthase prevented neuronal apoptosis. Further, SA led to concerted S-nitrosylation of proteins significantly associated with synaptic vesicle recycling and acetyl-CoA homeostasis. Our findings show that low-dose chronic exposure (0.1-1 ppm) to SA in the drinking water of mice led to S-nitrosylation of proteomic cysteines. Subsequent removal of arsenic from the drinking water reversed the biochemical alterations. CONCLUSIONS: This work develops a mechanistic understanding of the role of NO in arsenic-mediated toxicity in the brain, incorporating Ca2+ release and S-nitrosylation as important modifiers of neuronal protein function.

Chemoproteomics of matrix metalloproteases in a model of cartilage degeneration suggests functional biomarkers associated with posttraumatic osteoarthritis.

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity.

Analysis of an Integrated Human Multiorgan Microphysiological System for Combined Tolcapone Metabolism and Brain Metabolomics.

Human-on-a-chip systems are rapidly advancing due to the availability of human stem cells from a variety of tissues, but publications have utilized mostly simple methods of biochemical analysis. Here, we apply mass spectrometry to a sophisticated multiorgan human-on-a-chip system for the comprehensive study of tolcapone metabolite profiling and metabolomics. The developed human-on-a-chip includes seven interacting microphysiological systems (MPSs), brain, pancreas, liver, lung, heart, gut, and endometrium, with a mixer chamber for systemic circulation and tolcapone dose. We investigated tolcapone metabolism by analyzing the circulating medium using mass spectrometry. Twelve tolcapone metabolites were identified, three of which are newly reported. These metabolites demonstrated that oxidation, reduction, and conjugation reactions were the most important routes of tolcapone metabolism. In parallel, metabolomics in brain MPS evaluated the tolcapone influences on endogenous pathways in human brain. Untargeted metabolomics identified 18 key biomarkers significantly changed in human brain MPS after tolcapone dosing, which were mainly associated with perturbation of tryptophan and phenylalanine metabolism (BH4 cycle), glycerophospholipid metabolism, energy metabolism, and aspartate metabolism. This is the first example of successfully combining drug metabolism, metabolomics, and cell engineering to capture complex human physiology and the multiorgan interactions; the results we present here could be a step toward using analytical chemistry to advance the utilization of human-on-a-chip for testing both drug efficacy and toxicity in a single system.

S-nitrosation of proteins relevant to Alzheimer's disease during early stages of neurodegeneration.

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.

Magnetic silica nanoparticles for use in matrix-assisted laser desorption ionization mass spectrometry of labile biomolecules such as oligosaccharides, amino acids, peptides and nucleosides.

Magnetic silica nanoparticles (MSNPs) were prepared and applied for the first time as a matrix in MALDI MS for analysis of small thermally labile biomolecules including oligosaccharides, amino acids, peptides, nucleosides, and ginsenosides. The matrix was characterized by scanning electron microscopy and UV-vis spectroscopy. It displays good performance in analyses of such biomolecules in the positive ion mode. In addition, the method generates significantly less energetic ions compared to the use of carbon nanotubes or graphene-assisted LDI MS and thus produces intact molecular ions with little or no fragmentation. In addition, the MSNPs have better surface homogeneity and better salt tolerance and cause lower noise. It is assumed that the soft ionization observed when using MSNPs as a matrix is due to the specific surface area and the homogenous surface without large clusters. The matrices were applied to the unambiguous identification and relative quantitation of the water extract of Panax ginseng roots. Any false-positive results as obtained when using graphene and carbon nanotubes as a matrix were not observed. Graphical abstract Schematic presentation of the application of magnetic silica nanoparticles in laser desorption ionization mass spectrometry. Their use results in little or no fragmentation during analysis of small labile biomolecules with some advantages such as better surface homogeneity, high salt tolerance, and lower noise.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1ß, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1ß, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model.

The emergence of microphysiologic epithelial lung models using human cells in a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug development and to improve predictive power regarding in vivo drug clearance. In this study, an in vitro model of the airway comprising human primary lung epithelial cells cultured in a microfluidic platform was used to establish a physiologic state and to observe metabolic changes as a function of glucocorticoid exposure. Evaluation of mucus production rate and barrier function, along with lung-specific markers, demonstrated that the lungs maintained a differentiated phenotype. Initial concentrations of 100 nM hydrocortisone (HC) and 30 nM cortisone (C) were used to evaluate drug clearance and metabolite production. Measurements made using ultra-high-performance liquid chromatography and high-mass-accuracy mass spectrometry indicated that HC metabolism resulted in the production of C and dihydrocortisone (diHC). When the airway model was exposed to C, diHC was identified; however, no conversion to HC was observed. Multicompartmental modeling was used to characterize the lung bioreactor data, and pharmacokinetic parameters, including elimination clearance and elimination half-life, were estimated. Polymerse chain reaction data confirmed overexpression of 11-ß hydroxysteroid dehydrogenase 2 (11ßHSD2) over 11ßHSD1, which is biologically relevant to human lung. Faster metabolism was observed relative to a static model on elevated rates of C and diHC formation. Overall, our results demonstrate that this lung airway model has been successfully developed and could interact with other human tissues in vitro to better predict in vivo drug behavior.

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease.

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2'-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused three-dimensional human liver bioreactor.

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a three-dimensional human microphysiological hepatocyte-Kupffer cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and was assessed by the release of proinflammatory cytokines, interleukin 6 and tumor necrosis factor α. A sensitive and specific reversed-phase-ultra high-performance liquid chromatography-quadrupole time of flight-mass spectrometry method was used to evaluate hydrocortisone disappearance and metabolism at near physiologic levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for 2 days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10% of the loss, and 45-52% consisted of phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life, rate of elimination, clearance, and area under the curve, were 23.03 hours, 0.03 hour(-1), 6.6 × 10(-5) l⋅hour(-1), and 1.03 (mg/l)*h, respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized, and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically relevant tool for investigating hepatic function in an inflamed liver.

Untargeted Proteomics and Systems-Based Mechanistic Investigation of Artesunate in Human Bronchial Epithelial Cells.

The antimalarial drug artesunate is a semisynthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua. It is hypothesized to attenuate allergic asthma via inhibition of multiple signaling pathways. We used a comprehensive approach to elucidate the mechanism of action of artesunate by designing a novel biotinylated dihydroartemisinin (BDHA) to identify cellular protein targets of this anti-inflammatory drug. By adopting an untargeted proteomics approach, we demonstrated that artesunate may exert its protective anti-inflammatory effects via direct interaction with multiple proteins, most importantly with a number of mitochondrial enzymes related to glucose and energy metabolism, along with mRNA and gene expression, ribosomal regulation, stress responses, and structural proteins. In addition, the modulatory effects of artesunate on various cellular transcription factors were investigated using a transcription factor array, which revealed that artesunate can simultaneously modulate multiple nuclear transcription factors related to several major pro- and anti-inflammatory signaling cascades in human bronchial epithelial cells. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2), a key promoter of antioxidant mechanisms, which is inhibited by the Kelch-like ECH-associated protein 1 (Keap1). Our results demonstrate that, like other electrophilic Nrf2 regulators, artesunate activates this system via direct molecular interaction/modification of Keap1, freeing Nrf2 for transcriptional activity. Altogether, the molecular interactions and modulation of nuclear transcription factors provide invaluable insights into the broad pharmacological actions of artesunate in inflammatory lung diseases and related inflammatory disorders.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer.

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Gut microbiome phenotypes driven by host genetics affect arsenic metabolism.

Large individual differences in susceptibility to arsenic-induced diseases are well-documented and frequently associated with different patterns of arsenic metabolism. In this context, the role of the gut microbiome in directly metabolizing arsenic and triggering systemic responses in diverse organs raises the possibility that gut microbiome phenotypes affect the spectrum of metabolized arsenic species. However, it remains unclear how host genetics and the gut microbiome interact to affect the biotransformation of arsenic. Using an integrated approach combining 16S rRNA gene sequencing and HPLC-ICP-MS arsenic speciation, we demonstrate that IL-10 gene knockout leads to a significant taxonomic change of the gut microbiome, which in turn substantially affects arsenic metabolism.

Site-specific and redox-controlled S-nitrosation of thioredoxin.

Protein S-nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-nitrosation. Prior reports have studied thermodynamic end points of nitrosation reactions; however, the kinetics of Trx nitrosation has not previously been investigated. Using the transnitrosation agent, S-nitrosoglutathione, a kinetic analysis of the selectivity and redox dependence of Trx nitrosation at physiologically relevant concentrations and times was performed, utilizing a mass spectrometry-based method for the direct analysis of the nitrosated Trx. Reduced Trx (rTrx) was nitrosated 2.7-times faster than oxidized Trx (oTrx), and rTrx was nitrosated selectively on Cys62, whereas oTrx was nitrosated only on Cys73. These sites of nitrosation were confirmed at the peptide level using a novel modification of the biotin-switch technique called the reductive switch. These results suggest separate signaling pathways for Trx-SNO under different cellular redox states.

Mechanism-based triarylphosphine-ester probes for capture of endogenous RSNOs.

Nitrosothiols (RSNOs) have been proposed as important intermediates in nitric oxide (NO(•)) metabolism, storage, and transport as well as mediators in numerous NO-signaling pathways. RSNO levels are finely regulated, and dysregulation is associated with the etiology of several pathologies. Current methods for RSNO quantification depend on indirect assays that limit their overall specificity and reliability. Recent developments of phosphine-based chemical probes constitute a promising approach for the direct detection of RSNOs. We report here results from a detailed mechanistic and kinetic study for trapping RSNOs by three distinct phosphine probes, including structural identification of novel intermediates and stability studies under physiological conditions. We further show that a triarylphosphine-thiophenyl ester can be used in the absolute quantification of endogenous GSNO in several cancer cell lines, while retaining the elements of the SNO functional group, using an LC-MS-based assay. Finally, we demonstrate that a common product ion (m/z = 309.0), derived from phosphine-RSNO adducts, can be used for the detection of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological samples. Collectively, these findings establish a platform for the phosphine ligation-based, specific and direct detection of RSNOs in biological samples, a powerful tool for expanding the knowledge of the biology and chemistry of NO(•)-mediated phenomena.

Tunable membranes for free-flow zone electrophoresis in PDMS microchip using guided self-assembly of silica microbeads.

In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3-6 µm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a given pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be minimized by using ion selectivity of hydrogel-infiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity.

Comparative analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and γ-radiation.

Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.

Gut microbiome perturbations induced by bacterial infection affect arsenic biotransformation.

Exposure to arsenic affects large human populations worldwide and has been associated with a long list of human diseases, including skin, bladder, lung, and liver cancers, diabetes, and cardiovascular disorders. In addition, there are large individual differences in susceptibility to arsenic-induced diseases, which are frequently associated with different patterns of arsenic metabolism. Several underlying mechanisms, such as genetic polymorphisms and epigenetics, have been proposed, as these factors closely impact the individuals' capacity to metabolize arsenic. In this context, the role of the gut microbiome in directly metabolizing arsenic and triggering systemic responses in diverse organs raises the possibility that perturbations of the gut microbial communities affect the spectrum of metabolized arsenic species and subsequent toxicological effects. In this study, we used an animal model with an altered gut microbiome induced by bacterial infection, 16S rRNA gene sequencing, and inductively coupled plasma mass spectrometry-based arsenic speciation to examine the effect of gut microbiome perturbations on the biotransformation of arsenic. Metagenomics sequencing revealed that bacterial infection significantly perturbed the gut microbiome composition in C57BL/6 mice, which in turn resulted in altered spectra of arsenic metabolites in urine, with inorganic arsenic species and methylated and thiolated arsenic being perturbed. These data clearly illustrated that gut microbiome phenotypes significantly affected arsenic metabolic reactions, including reduction, methylation, and thiolation. These findings improve our understanding of how infectious diseases and environmental exposure interact and may also provide novel insight regarding the gut microbiome composition as a new risk factor of individual susceptibility to environmental chemicals.

A filtered database search algorithm for endogenous serum protein carbonyl modifications in a mouse model of inflammation.

During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum.