Quantitative Nontarget Analysis of CECs in Environmental Samples Can Be Improved by Considering All Mass Adducts.

Quantitative nontarget analysis (qNTA) for liquid chromatography coupled to high-resolution mass spectrometry enables a more comprehensive assessment of environmental samples. Previous studies have shown that correlations between a compound's ionization efficiency and a range of molecular descriptors can predict the compound's concentration within a factor of 5. In this study, the qNTA approach was further improved by considering all mass adducts instead of only the protonated ion. The model was based on a quantitative structure-property relationship (QSPR), including 216 contaminants of emerging concern (CECs), of which 80 exhibited adduct formation that accounted for >10% of the total peak intensity. When all mass adducts were included, the test set coefficient of determination improved to Q2 = 0.855 compared to Q2 = 0.670 when only the protonated ions were considered (test set median RF error factor 1.6). The inclusion of all adducts was also important to transfer the RF QSPR model reliably. It was assumed that RF variations are sequence-dependent; therefore, a second QSPR model for the prediction of the transferability factor was built for each sequence. For validation, samples were analyzed up to two years apart. The median prediction fold change was 1.74 for analytical standards (63 compounds) and 2.4 for enriched wastewater effluent samples (41 compounds), with 80% of the compounds predicted within a fold change of 2.4 and 3.3, respectively. The model was also validated on a second instrument, where 80% of the 26 compounds in wastewater effluent were predicted within a factor of 3.8.

Correction of Matrix Effects for Reliable Non-target Screening LC-ESI-MS Analysis of Wastewater.

Matrix effects are well-known challenges for accurate and comparable measurements with liquid chromatography (LC) electrospray ionization mass spectrometry (ESI-MS). This study describes a three-step method to evaluate and compensate for matrix effects in enriched wastewater extracts using LC ESI-high-resolution MS (HRMS). As a first step, the "dilute and shoot" approach was used to determine the optimal relative enrichment factor (REF) for a direct comparison between wastewater influent (REF 10) and effluent (REF 50) extracts. However, the rapid decrease in the number of non-target compounds detected with increasing dilution leads to the need for a correction of the matrix effect for analyzing samples with higher REFs. As a second step, the observed matrix effect at higher REFs was corrected by the retention time-dependent matrix effect. A new scaling (TiChri scale) of the matrix effect was introduced, which demonstrates that the total ion chromatogram (TIC) predicts the matrix effect as effectively as post-column infusion (PCI) approaches; thus, the average median matrix effect was improved from -65 to 1% for influent (REF 100) and from -46 to -2% for effluent extracts (REF 250). The TIC traces for concentrated (REF 250) influent and effluent samples were successfully used to correct the matrix effects and allowed the extent of micropollutant degradation in three WWTPs to be quantified. As a final step, the residual structure-specific matrix effect was predicted and corrected by quantitative structure-property relationships (QSPR), which led to a further correction of the matrix effect to 0 ± 7% for 65 compounds.

Isolate-Specific Effect of Entomopathogenic Endophytic Fungi on Population Growth of Two-Spotted Spider Mite (Tetranychus urticae Koch) and Levels of Steroidal Glycoalkaloids in Tomato.

Entomopathogenic fungi (EPF) can be experimentally established in several plant species as endophytes. Ecological effects of EPF inoculations on plant growth and plant-herbivore interactions have been demonstrated, potentially by altering plant physiological responses. However, the role of these responses in plant-fungus-herbivore tripartite interactions has not been well elucidated. Steroidal glycoalkaloids (SGAs) are plant specialized metabolites with bioactive properties against arthropod herbivores. Here, the effects of seed treatments by three EPF isolates, representing Beauveria bassiana, Metarhizium brunneum, and M. robertsii, on population growth of two-spotted spider mites (Tetranychus urticae Koch) were evaluated on tomato (Solanum lycopersicum). The levels of two SGAs, α-tomatine and dehydrotomatine, were determined in tomato leaves by LC-MS with and without T. urticae infestations after EPF inoculations. Interestingly, the population growth of T. urticae was significantly highest with M. brunneum and lowest with M. robertsii and B. bassiana at 15 days after infestation. Overall there was a significant negative correlation between SGAs content and the number of T. urticae. The levels of SGAs were significantly induced by T. urticae presence in all treatments, while only M. robertsii showed significantly higher levels of SGAs than M. brunneum and control in one of two experiments. Contrastingly, the effects on SGAs accumulation and population growth of T. urticae did not directly correlate with EPF endophytic colonization patterns of the inoculated plants. This study suggests a link between ecological effects and physiological responses mediated by EPF inoculations and T. urticae infestation with potential implications for plant protection.

Carnosine and Carcinine Derivatives Rapidly React with Hypochlorous Acid to Form Chloramines and Dichloramines.

Hypochlorous acid (HOCl) is a highly reactive, toxic species generated by neutrophils via the action of myeloperoxidase in order to destroy invading pathogens. However, when HOCl is produced inappropriately, it can damage host tissue and proteins and plays a role in the initiation and progression of disease. Carnosine, a peptide of ß-alanine and histidine, has been shown to react rapidly with HOCl yielding monochloramines and can undergo intramolecular transchlorination. The current study examines the kinetics and pH dependence of the reactions of carnosine and novel structural derivatives with HOCl and the occurrence of intra- and intermolecular transchlorination processes. We demonstrate that the transchlorination reactions of carnosine are pH dependent, with intramolecular transfer favored at higher pH. Carcinine, having a structure identical to carnosine though lacking the carboxylic acid group of the histidine residue, reacts with HOCl and forms monochloramines though intramolecular transfer reactions are not observed, and this is supported by computational modeling. Novel analogues with one (carnosine+1) and two (carnosine+2) methylene groups in the alkyl chain of the ß-alanine react with HOCl to yield monochloramines that undergo transchlorinations to yield a mixture of mono- and dichloramines. The latter are stable over 24 h. The ability of carnosine and derivatives to react rapidly with HOCl to give long-lived, poorly reactive, species may prevent damage to proteins and other targets at sites of inflammation.

Tryptophan oxidation in proteins exposed to thiocyanate-derived oxidants.

Human defensive peroxidases, including lactoperoxidase (LPO) and myeloperoxidase (MPO), are capable of catalyzing the oxidation of halides (X(-)) by H2O2 to give hypohalous acids (HOX) for the purpose of cellular defense. Substrate selectivity depends upon the relative abundance of the halides, but the pseudo-halide thiocyanate (SCN(-)) is a major substrate, and sometimes the exclusive substrate, of all defensive peroxidases in most physiologic fluids. The resulting hypothiocyanous acid (HOSCN) has been implicated in cellular damage via thiol oxidation. While thiols are believed to be the primary target of HOSCN in vivo, Trp residues have also been implicated as targets for HOSCN. However, the mechanism involved in HOSCN-mediated Trp oxidation was not established. Trp residues in proteins appeared to be susceptible to oxidation by HOSCN, whereas free Trp and Trp residues in small peptides were found to be unreactive. We show that HOSCN-induced Trp oxidation is dependent on pH, with oxidation of free Trp, and Trp-containing peptides observed when the pH is below 2. These conditions mimic those employed previously to precipitate proteins after treatment with HOSCN, which accounts for the discrepancy in the results reported for proteins versus free Trp and small peptides. The reactant in these cases may be thiocyanogen ((SCN)2), which is produced by comproportionation of HOSCN and SCN(-) at low pH. Reaction of thiocyanate-derived oxidants with protein Trp residues at low pH results in the formation of a number of oxidation products, including mono- and di-oxygenated derivatives, which are also formed with other hypohalous acids. Our data suggest that significant modification of Trp by HOSCN in vivo is likely to have limited biological relevance.

Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctional high-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorous acid (HOCl).

Oxidative modification of HDLs (high-density lipoproteins) by MPO (myeloperoxidase) compromises its anti-atherogenic properties, which may contribute to the development of atherosclerosis. Although it has been established that HOCl (hypochlorous acid) produced by MPO targets apoA-I (apolipoprotein A-I), the major apolipoprotein of HDLs, the role of the other major oxidant generated by MPO, HOSCN (hypothiocyanous acid), in the generation of dysfunctional HDLs has not been examined. In the present study, we characterize the structural and functional modifications of lipid-free apoA-I and rHDL (reconstituted discoidal HDL) containing apoA-I complexed with phospholipid, induced by HOSCN and its decomposition product, OCN- (cyanate). Treatment of apoA-I with HOSCN resulted in the oxidation of tryptophan residues, whereas OCN- induced carbamylation of lysine residues to yield homocitrulline. Tryptophan residues were more readily oxidized on apoA-I contained in rHDLs. Exposure of lipid-free apoA-I to HOSCN and OCN- significantly reduced the extent of cholesterol efflux from cholesterol-loaded macrophages when compared with unmodified apoA-I. In contrast, HOSCN did not affect the anti-inflammatory properties of rHDL. The ability of HOSCN to impair apoA-I-mediated cholesterol efflux may contribute to the development of atherosclerosis, particularly in smokers who have high plasma levels of SCN- (thiocyanate).

Redox factors in the antioxidant activity of nitroxides toward DNA guanyl and 2-deoxyribose-peroxyl radicals.

A series of eight nitroxide compounds (four substituted piperidines, three pyrrolidines and one oxo-piperidine) are found to undergo electron transfer to 2'-deoxyribose-peroxyl and the guanyl radical. One-electron oxidation potentials of the nitroxides to oxoammonium cations (oxoammonium reduction potential), E0', have been measured against a common redox indicator, chlorpromazine, and found to span the range 751 ± 15 mV to 973 ± 15 mV. Fast chemical reduction of the 2'-deoxyribose-peroxyl radical to the hydroperoxide, generated by •OH radical attack on 2-deoxyribose, dR, in oxygenated aqueous solution, is a redox-dependent reaction, with rate constants of 0.8-3.5 x 107 M-1 s-1.The guanyl radicals, produced upon one-electron oxidation of 2'-deoxyguanosine monophosphate, dG, by the selenite radical, SeO3•-, react with the nitroxides in a redox-independent reaction with diffusion rate constants of 1-2 x 108 M-1 s-1. These findings represent a possible antioxidant role for nitroxides in the fast chemical repair of DNA radicals, which is supported by an in vitro strand break study using a plasmid.

Comprehensive two-dimensional gas chromatography as a tool for targeted and non-targeted analysis of contaminants of emerging concern in wastewater.

Wastewater is a major reservoir for chemical contaminants, both anthropogenic and biogenic. Recent chemical and toxicological analysis reveals the abundance and impact of these compounds, often termed contaminants of emerging concern (CECs). Concurrently, incomplete removal of these compounds in wastewater treatment plants sets a precedent for detailed characterisation and monitoring of such substances. Although liquid chromatography (LC) is frequently used for analysis of CECs in wastewater, gas chromatography (GC) maintains its significance for non-polar to mid-polar analytes. GC offers advantages such as increased separation efficiency, fewer matrix effects, and greater availability and reliability of reference mass spectra compared to LC. Comprehensive two-dimensional gas chromatography (GC × GC) delivers unmatched peak capacity and separational capabilities, critical in the resolution of diverse compound groups present within wastewater. When coupled with high resolution mass spectrometry, it provides a powerful identification tool with spectral databases and both 1st and 2nd dimensional retention indices, and has allowed for the separation, reliable annotation and characterisation of diverse CECs within wastewater in recent years. Herein, on the basis of recent studies from the last fifteen years, we outline cutting-edge methodologies and strategies for wastewater analysis using GC × GC. This includes sample preparation, derivatization of polar analytes, instrumental setup, and data analysis, ultimately providing the reader a framework for future non-targeted analysis of wastewater and other complex environmental matrices.

System stability and signal enhancement with analyte protectants: Gas chromatography analysis of oxygenated-polycyclic aromatic hydrocarbons.

Matrix effects can affect detection limits, precision, and accuracy and lead to signal enhancement or suppression effects in gas chromatography analysis. Analyte protectants, such as shikimic acid and gluconolactone, can imitate the effect of matrix components and reduce the differences in matrix effect between samples. This study aimed to investigate the ability of analyte protectants to enhance gas chromatography detector signals of different oxygenated-polycyclic aromatic hydrocarbons. Addition of 100 µg L-1 shikimic acid and 200 µg L-1 gluconolactone effectively enhanced detector response of the investigated target compounds. Addition of a higher content of analyte protectants did not result in any further enhancement. It was found that between four and eleven consecutive injections of a standard solution with analyte protectants were required to obtain a stable compound response. The long-term signal stability was then maintained with subsequent injections, though an overall negative drift of the system was observed over the sequence of 200 investigated injections. Analysis of the actual sample matrix instead of standards in pure solvent, as presented in this study, could also be a way to minimize the required number of injections. Shikimic acid and gluconolactone were first and foremost able to enhance signals of oxygenated-polycyclic aromatic hydrocarbons with similar functional groups (hydroxyl) in their molecular structure. It can be relevant to consider alternative analyte protectants with different functional groups according to the type of target compounds investigated.

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage.

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

Computational design of effective, bioinspired HOCl antioxidants: the role of intramolecular Cl+ and H+ shifts.

The enzyme myeloperoxidase generates significant amounts of hypochlorous acid (HOCl) at sites of inflammation to inflict oxidative damage upon invading pathogens. However, excessive production of this potent oxidant is associated with numerous inflammatory diseases. Recent kinetic measurements suggest that the endogenous antioxidant carnosine is an effective HOCl scavenger. On the basis of computational modeling, we suggest a possible mechanism for this antioxidant activity. We find that a unique structural relationship between three adjacent functional groups (imidazole, carboxylic acid, and terminal amine) enables an intramolecular chlorine transfer to occur. In particular, two sequential proton shifts are coupled with a Cl(+) shift converting the kinetically favored product (chlorinated at the imidazole nitrogen) into the thermodynamically favored product (chlorinated at the terminal amine) effectively trapping the chlorine. We proceed to design systems that share similar structural features to those of carnosine but with even greater HOCl-scavenging capabilities.

Preventing protein oxidation with sugars: scavenging of hypohalous acids by 5-selenopyranose and 4-selenofuranose derivatives.

Heme peroxidases including myeloperoxidase (MPO) are released at sites of inflammation by activated leukocytes. MPO generates hypohalous acids (HOX, X = Cl, Br, SCN) from H(2)O(2); these oxidants are bactericidal and are key components of the inflammatory response. However, excessive, misplaced or mistimed production can result in host tissue damage, with this implicated in multiple inflammatory diseases. We report here methods for the conversion of simple monosaccharide sugars into selenium- and sulfur-containing species that may act as potent water-soluble scavengers of HOX. Competition kinetic studies show that the seleno species react with HOCl with rate constants in the range 0.8-1.0 × 10(8) M(-1) s(-1), only marginally slower than those for the most susceptible biological targets including the endogenous antioxidant, glutathione. The rate constants for the corresponding sulfur-sugars are considerably slower (1.4-1.9 × 10(6) M(-1) s(-1)). Rate constants for reaction of the seleno-sugars with HOBr are ~8 times lower than those for HOCl (1.0-1.5 × 10(7) M(-1) s(-1)). These values show little variation with differing sugar structures. Reaction with HOSCN is slower (~10(2) M(-1) s(-1)). The seleno-sugars decreased the extent of HOCl-mediated oxidation of Met, His, Trp, Lys, and Tyr residues, and 3-chlorotyrosine formation, on both isolated bovine serum albumin and human plasma proteins, at concentrations as low as 50 µM. These studies demonstrate that novel selenium (and to a lesser extent, sulfur) derivatives of monosaccharides could be potent modulators of peroxidase-mediated damage at sites of acute and chronic inflammation, and in multiple human pathologies.

Photo-oxidation of proteins.

Photo-induced damage to proteins occurs via multiple pathways. Direct damage induced by UVB (λ 280-320 nm) and UVA radiation (λ 320-400 nm) is limited to a small number of amino acid residues, principally tryptophan (Trp), tyrosine (Tyr), histidine (His) and disulfide (cystine) residues, with this occurring via both excited state species and radicals. Indirect protein damage can occur via singlet oxygen ((1)O(2)(1)Δ(g)), with this resulting in damage to Trp, Tyr, His, cystine, cysteine (Cys) and methionine (Met) residues. Although initial damage is limited to these residues multiple secondary processes, that occur both during and after radiation exposure, can result in damage to other intra- and inter-molecular sites. Secondary damage can arise via radicals (e.g. Trp, Tyr and Cys radicals), from reactive intermediates generated by (1)O(2) (e.g. Trp, Tyr and His peroxides) and via molecular reactions of photo-products (e.g. reactive carbonyls). These processes can result in protein fragmentation, aggregation, altered physical and chemical properties (e.g. hydrophobicity and charge) and modulated biological turnover. Accumulating evidence implicates these events in cellular and tissue dysfunction (e.g. apoptosis, necrosis and altered cell signaling), and multiple human pathologies.

Second dimension retention indices in "normal" orthogonality comprehensive two-dimensional gas chromatography using single standard injection generated isovolatility curves.

This work builds upon recent developments in the field of second dimensional (2D) retention indices (RI) for use in comprehensive two-dimensional gas chromatography (GC×GC), expanding application to the most commonly used "normal" orthogonality column configuration, where 2D RI are rarely employed. Initially, one dimensional retention indices for 80 wastewater pollutants were determined by GC-MS on a mid-polar ZB-50 column. In order to determine the 2D RIs for peaks detected in wastewater extracts separated by GC×GC -MS, a single injection of a ten-compound standard mix allowed the construction of model-generated isovolatility curves. These curves were used for the determination of 2D RIs of compounds initially identified on the basis of the mass spectral match factor and 1D RIs. Good agreements (average deviation of 1.7%) were observed between the calculated 2D RIs and the measured reference RIs for these compounds. These results show that this approach provides an additional level of confidence for the identification of compounds detected in GC×GC-MS and demonstrates the potential of this approach for improved compound identification in non-targeted analysis.

Ancient Biosyntheses in an Oil Crop: Glucosinolate Profiles in Limnanthes alba and Its Relatives (Limnanthaceae, Brassicales).

The glucosinolate (GSL) profiles of four Limnanthaceae species, including the oil crop Limnanthes alba (meadowfoam), were investigated by an ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QToF-MS/MS) analysis of desulfoGSLs after desulfation of native GSLs, supplemented by NMR of desulfated 2-hydroxy-2-methylpropylGSL and 3-methoxybenzylGSL. Leaves, roots, and seeds were investigated, providing an overview of biosynthetic capabilities in the genera Floerkea and Limnanthes. Methoxyl groups on benzylGSLs were in meta but not para positions; two 3,5-disubstituted benzylGSLs are tentatively proposed. 2-Hydroxy-2-methylpropylGSL was accompanied by an isomer that was not a previously reported GSL. The combined GSL profile of the family included GSLs derived from valine, leucine, isoleucine, phenylalanine, and tyrosine, and possibly methionine and tryptophan. Substituted indole GSLs and GSLs derived from chain-elongated amino acids or alanine were searched for but not detected. Hypothetic glycosides of GSLs were detected at low levels. Based on biochemical interpretation, we suggest biosynthetic schemes and gene families (CYP79C, GSOH) relevant for tailoring GSL profiles in Limnanthes crops.

One-electron reduction of N-chlorinated and N-brominated species is a source of radicals and bromine atom formation.

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong bactericidal oxidants that are generated by the human immune system but are implicated in the development of many human inflammatory diseases (e.g., atherosclerosis, asthma). These oxidants react readily with sulfur- and nitrogen-containing nucleophiles, with the latter generating N-halogenated species (e.g., chloramines/bromamines (RR'NX; X = Cl, Br)) as initial products. Redox-active metal ions and superoxide radicals (O(2)(•-)) can reduce N-halogenated species to nitrogen- and carbon-centered radicals. N-Halogenated species and O(2)(•-) are generated simultaneously at sites of inflammation, but the significance of their interactions remains unclear. In the present study, rate constants for the reduction of N-halogenated amines, amides, and imides to model potential biological substrates have been determined. Hydrated electrons reduce these species with k(2) > 10(9) M(-1) s(-1), whereas O(2)(•-) reduced only N-halogenated imides with complex kinetics indicative of chain reactions. For N-bromoimides, heterolytic cleavage of the N-Br bond yielded bromine atoms (Br(•)), whereas for other substrates, N-centered radicals and Cl(-)/Br(-) were produced. High-level quantum chemical procedures have been used to calculate gas-phase electron affinities and aqueous solution reduction potentials. The effects of substituents on the electron affinities of aminyl, amidyl, and imidyl radicals are rationalized on the basis of differential effects on the stabilities of the radicals and anions. The calculated reduction potentials are consistent with the experimental observations, with Br(•) production predicted for N-bromosuccinimide, while halide ion formation is predicted in all other cases. These data suggest that interaction of N-halogenated species with O(2)(•-) may produce deleterious N-centered radicals and Br(•).

Cascading Effects of Root Microbial Symbiosis on the Development and Metabolome of the Insect Herbivore Manduca sexta L.

Root mutualistic microbes can modulate the production of plant secondary metabolites affecting plant-herbivore interactions. Still, the main mechanisms underlying the impact of root mutualists on herbivore performance remain ambiguous. In particular, little is known about how changes in the plant metabolome induced by root mutualists affect the insect metabolome and post-larval development. By using bioassays with tomato plants (Solanum lycopersicum), we analyzed the impact of the arbuscular mycorrhizal fungus Rhizophagus irregularis and the growth-promoting fungus Trichoderma harzianum on the plant interaction with the specialist insect herbivore Manduca sexta. We found that root colonization by the mutualistic microbes impaired insect development, including metamorphosis. By using untargeted metabolomics, we found that root colonization by the mutualistic microbes altered the secondary metabolism of tomato shoots, leading to enhanced levels of steroidal glycoalkaloids. Untargeted metabolomics further revealed that root colonization by the mutualists affected the metabolome of the herbivore, leading to an enhanced accumulation of steroidal glycoalkaloids and altered patterns of fatty acid amides and carnitine-derived metabolites. Our results indicate that the changes in the shoot metabolome triggered by root mutualistic microbes can cascade up altering the metabolome of the insects feeding on the colonized plants, thus affecting the insect development.

Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale.

Ability of hypochlorous acid and N-chloramines to chlorinate DNA and its constituents.

Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal properties and plays an important role in the human immune system, this oxidant also causes damage to tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Nalpha-acetyl-histidine (His-C), Nalpha-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and His-C resulted in the formation of 5-chloro-2'-deoxycytidine (5CldC), 8-chloro-2'-deoxyadenosine (8CldA) and 8-chloro-2'-deoxyguanosine (8CldG). With the nucleosides, 8CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers.