Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Antineoplastic effects of cassava-cyanide extract on human glioblastoma (LN229) cells.

Several natural compounds reduce tumour cell growth and metastasis by inducing programmed cell death. Cassava (Manihot esculenta Crantz) contains cyanogenic glycosides such as, linamarin and lotaustralin, can be enzymatically cleaved by linamarase to release hydrogen cyanide (HCN), which can have therapeutic benefits against hypertension, asthma, and cancer. We have developed a technology for isolating bio-active principles from cassava leaves.The present study is designed to analyze the cytotoxic effect of cassava cyanide extract (CCE) against human glioblastoma cells (LN229). The treatment of CCE demonstrated a dose dependent toxicity on glioblastoma cells. At higher concentration tested, the CCE (400 µg/mL) was found to be cytotoxic, reducing the cell viability to 14.07 ± 2.15% by negatively influencing the mitochondrial activity, and lysosomal and cytoskeletal integrity. Coomassie's brilliant blue staining confirmed cells' morphological aberration after 24 h of treatment with CCE. Moreover, DCFH-DA assay and Griess reagent showed an increase in ROS but a decrease in RNS production at a concentration of CCE. Flow cytometry analysis revealed that CCE interfered with G0/G1, S, and G2/M stages of the cell cycle of glioblastoma, and Annexin/PI staining indicated a dose-dependent increase in cell death, confirming the toxic nature of CCE on LN229 cells. These findings suggest that cassava cyanide extract has potential as an antineoplastic agent against glioblastoma cells, which is an aggressive and difficult-to-treat type of brain cancer. However, it is important to note that the study was conducted in vitro, and further research is necessary to assess the safety and efficacy of CCE in vivo. Additionally, it is essential to establish the optimal dose and potential side effects before considering its use as a therapeutic agent.

Co-opted genes of algal origin protect C. elegans against cyanogenic toxins.

Amygdalin is a cyanogenic glycoside enriched in the tissues of many edible plants, including seeds of stone fruits such as cherry (Prunus avium), peach (Prunus persica), and apple (Malus domestica). These plants biosynthesize amygdalin in defense against herbivore animals, as amygdalin generates poisonous cyanide upon plant tissue destruction.1,2,3,4 Poisonous to many animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals.5 Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1, and cysl-2. A screen of a natural product library for hypoxia-independent regulators of HIF-1 identifies amygdalin as a potent activator of cysl-2, a HIF-1 transcriptional target that encodes a cyanide detoxification enzyme in C. elegans. As a cysl-2 paralog similarly essential for amygdalin resistance, cysl-1 encodes a protein homologous to cysteine biosynthetic enzymes in bacteria and plants but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis indicates that cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Since acquisition, these two genes evolved division of labor in a cellular circuit to detect and detoxify cyanide. Thus, algae-to-nematode HGT and subsequent gene function co-option events may facilitate host survival and adaptation to adverse environmental stresses and biogenic toxins.

Gasotransmitter H2S accelerates seed germination via activating AOX mediated cyanide-resistant respiration pathway.

Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of L-cysteine desulfhydrase (EC 4.4.1.28, LCD and DES1) responsible for H2S production. Moreover, seed germination could be significantly accelerated by exogenous NaHS (the H2S donor) fumigation and over-expressing DES1, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. We also confirmed that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition. Furthermore, exogenous H2S fumigation and over-expressing DES1 could significantly reinforced imbibition induced increase of both the AOX1A expression and AOX protein abundance, while this increase could be obviously weakened in lcd/des1. Additionally, exogenous H2S fumigation mediated post-translational modification to keep AOX in its reduced and active state, which might involve H2S induced improvement of the reduced GSH content and the cell reducing power. The promotive effect of H2S on germination was clearly impaired by inducing aox1a mutation, indicating that AOX acts downstream of H2S signaling to accelerate seed germination. Consequently, H2S signaling was activated during germination then acted as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide-resistant respiration, providing evidence for more extensive studies of H2S signaling.

Structural and functional characterization of ß-cyanoalanine synthase from Tetranychus urticae.

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the ß-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in ß-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of ß-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of ß-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.

Discovery of Sortase A covalent inhibitors with benzofuranene cyanide structures as potential antibacterial agents against Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase of most gram-positive bacteria that is responsible for the anchoring of many surface protein virulence factors to the cell wall. SrtA ablation has demonstrated to alleviate the infection without affecting the viability of bacteria. Herein, a series of benzofuran cyanide derivatives were synthesized and evaluated. Several compounds exhibited excellent inhibitory activity against SrtA with IC50 values from 3.3 µM to 21.8 µM compared with the known SrtA inhibitor pHMB (IC50 = 130 µM). Ⅲ-1, Ⅲ-15, Ⅲ-34 and V-1 showed potent inhibitory effects on biofilm formation with IC50 values from 2.1 µM to 54.2 µM. Invasion assays showed the four compounds caused a decrease of 4%-24.0% in the uptake of the S. aureus strain by 293T cells. Further assay showed that compound Ⅲ-15 decreased the amount of cell wall-associated protein A by 26.5%. Structure-activity relationship and docking studies demonstrated that the acrylonitrile moiety of the compounds played an important role in enhancing the activity. When the double bond of acrylonitrile changed to single bond, the activity was decreased significantly. This indicates that acrylonitrile, which is a Michael receptor, can inhibit the activity of SrtA by covalent binding effectively to the thiol group of Cys184.

Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2.

[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG-HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics.

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H2S and may be considered the fourth mammalian gasotransmitter.

Photodynamics of Asymmetric Di-Iron-Cyano Hydrogenases Examined by Time-Resolved Mid-Infrared Spectroscopy.

Two anionic asymmetric Fe-Fe hydrogenase model compounds containing a single cyano (CN) and five carboxyl (CO) ligands, [Et4N][Fe2(µ-S2C3H6)(CO)5(CN)1] and [Et4N][Fe2(µ-S2C2H4)(CO)5(CN)1], dissolved in room-temperature acetonitrile, are examined. The molecular asymmetry affects the redox potentials of the central iron atoms, thus changing the photophysics and possible catalytic properties of the compounds. Femtosecond ultraviolet excitation with mid-infrared probe spectroscopy of the model compounds was employed to better understand the ultrafast dynamics of the enzyme-active site. Continuous ultraviolet lamp excitation with Fourier transform infrared (FTIR) spectroscopy was also used to explore stable product formation on the second timescale. For both model compounds, two timescales are observed; a 20-30 ps decay and the formation of a long-lived photoproduct. The picosecond decay is assigned to vibrational cooling and rotational dynamics, while the residual spectra remain for up to 300 ps, suggesting the formation of new photoproducts. Static FTIR spectroscopy yielded a different stable photoproduct than that observed on the ultrafast timescale. Density functional theory calculations simulated photoproducts for CO-loss and CN-loss isomers, and the resulting photoproduct spectra suggest that the picosecond transients arise from a complex mixture of isomerization after CO-loss, while dimerization and formation of a CN-containing Fe-CO-Fe bridged species are also considered.

Aryl isocyanide derivative for one-pot synthesis of purification-free 99mTc-labeled hexavalent targeting probe.

INTRODUCTION: 99mTc-labeled hexavalent probes can be readily synthesized by the coordination of six equivalent isocyanide ligands towards TcI, and alkyl isocyanide ligands have been extensively used for preparing such probes. However, high ligand concentration (>1 mM) is generally required due to their insufficient coordination ability to TcI. METHODS AND RESULTS: In this study, we revealed that aryl isocyanide ligands, which have greater π-accepting ability compared with alkyl ones, provided 99mTc-labeled hexavalent probes in high radiochemical yields (>95%) even at low ligand concentration (50 µM). We applied this finding to the synthesis of a 99mTc-labeled hexavalent RGD probe, targeting integrin αvß3. This 99mTc-labeled probe was prepared in a 5 min reaction at ligand concentration of 50 µM, and exhibited high tumor localization in vivo without post-labeling purification. CONCLUSION: The present findings indicate that aryl isocyanide ligands would be a useful precursor to a variety of 99mTc-labeled hexavalent targeting probes for molecular imaging of saturable systems. ADVANCES IN KNOWLEDGE: Aryl isocyanide is a better precursor than alkyl isocyanide for preparing 99mTc-labeled hexavalent targeting probe. IMPLICATION FOR PATIENT CARE: This work provides a straightforward method to prepare molecular imaging agents of high target uptake, which would facilitate nuclear medicine imaging in clinical settings.

Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses.

The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.

Ethylene Biosynthesis Inhibition Combined with Cyanide Degradation Confer Resistance to Quinclorac in Echinochloa crus-galli var. mitis.

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of ß-cyanoalanine synthase (ß-CAS). Resistant biotypes exhibited higher ß-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced ß-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.

Root ethylene mediates rhizosphere microbial community reconstruction when chemically detecting cyanide produced by neighbouring plants.

BACKGROUND: Stress-induced hormones are essential for plants to modulate their microbiota and dynamically adjust to the environment. Despite the emphasis of the role of the phytohormone ethylene in the plant physiological response to heterospecific neighbour detection, less is known about how this activated signal mediates focal plant rhizosphere microbiota to enhance plant fitness. Here, using 3 years of peanut (Arachis hypogaea L.), a legume, and cyanide-containing cassava (Manihot esculenta Crantz) intercropping and peanut monocropping field, pot and hydroponic experiments in addition to exogenous ethylene application and soil incubation experiments, we found that ethylene, a cyanide-derived signal, is associated with the chemical identification of neighbouring cassava and the microbial re-assemblage in the peanut rhizosphere. RESULTS: Ethylene production in peanut roots can be triggered by cyanide production of neighbouring cassava plants. This gaseous signal alters the microbial composition and re-assembles the microbial co-occurrence network of peanut by shifting the abundance of an actinobacterial species, Catenulispora sp., which becomes a keystone in the intercropped peanut rhizosphere. The re-assembled rhizosphere microbiota provide more available nutrients to peanut roots and support seed production. CONCLUSIONS: Our findings suggest that root ethylene acts as a signal with a dual role. It plays a role in perceiving biochemical cues from interspecific neighbours, and also has a regulatory function in mediating the rhizosphere microbial assembly, thereby enhancing focal plant fitness by improving seed production. This discovery provides a promising direction to develop novel intercropping strategies for targeted manipulations of the rhizosphere microbiome through phytohormone signals. Video abstract.

Overexpressed ß-cyanoalanine synthase functions with alternative oxidase to improve tobacco resistance to salt stress by alleviating oxidative damage.

ß-Cyanoalanine synthase (ß-CAS) is an enzyme involved in cyanide detoxification. However, little information is available regarding the effects of ß-CAS activity changes on plant resistance to environmental stress. Here, we found that ß-CAS overexpression (CAS-OE) improves the resistance of tobacco plants to salt stress, whereas plants with ß-CAS silencing suffer more oxidative damage than wild-type plants. Notably, blocking respiration by the alternative oxidase (AOX) pathway significantly aggravates stress injury and impairs the salt stress tolerance mediated by CAS-OE. These findings present novel insights into the synergistic effect between ß-CAS and AOX in protecting plants from salt stress, where ß-CAS plays a vital role in restraining cyanide accumulation, and AOX helps to alleviate the toxic effect of cyanide.

Cyanide produced with ethylene by ACS and its incomplete detoxification by ß-CAS in mango inflorescence leads to malformation.

Malformation of mango inflorescences (MMI) disease causes severe economic losses worldwide. Present research investigates the underlying causes of MMI. Results revealed significantly higher levels of cyanide, a by-product of ethylene biosynthesis, in malformed inflorescences (MI) of mango cultivars. There was a significant rise in ACS transcripts, ACS enzyme activity and cyanide and ethylene levels in MI as compared to healthy inflorescences (HI). Significant differences in levels of methionine, phosphate, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, ascorbate and glutathione, and activities of dehydroascorbate reductase and glutathione reductase were seen in MI over HI. Further, a lower expression of ß-cyanoalanine synthase (ß-CAS) transcript was associated with decreased cellular ß-CAS activity in MI, indicating accumulation of unmetabolized cyanide. TEM studies showed increased gum-resinosis and necrotic cell organelles, which might be attributed to unmetabolized cyanide. In field trials, increased malformed-necrotic-inflorescence (MNI) by spraying ethrel and decreased MNI by treating with ethylene inhibitors (silver and cobalt ions) further confirmed the involvement of cyanide in MMI. Implying a role for cyanide in MMI at the physiological and molecular level, this study will contribute to better understanding of the etiology of mango inflorescence malformation, and also help manipulate mango varieties genetically for resistance to malformation.

Development of a UPLC-ESI-MS/MS method to measure urinary metabolites of selected VOCs: Benzene, cyanide, furfural, furfuryl alcohol, 5-hydroxymethylfurfural, and N-methyl-2-pyrrolidone.

We report on the development of an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously measuring eight biomarkers of volatile organic compound (VOC) exposure, with potential application to e-cigarette aerosol biomonitoring. Phenylmercapturic acid (PMA) and trans, trans-muconic acid (tt-MA) are metabolites of benzene; 2-aminothiazoline-4-carboxylic acid (ATCA) is a metabolite of cyanide; N-2-furoylglycine (N2FG) is a metabolite of furfural and furfuryl alcohol; 5-hydroxymethylfuroic acid (HMFA), 5-hydroxymethyl-2-furoylglycine (HMFG), and 2,5-furandicarboxylic acid (FDCA) are metabolites of 5-hydroxymethylfurfural; and 5-hydroxy-N-methylpyrrolidone (5HMP) is a metabolite of N-methyl-2-pyrrolidone. A pentafluorophenyl-modified silica column was used for chromatographic separation. The overall run time for the method is about 6 min per sample injection. The method has low to sub-nanograms per milliliter sensitivity, linearity over 3 orders of magnitude, and precision and accuracy within 15%. The method was used to measure human urine samples. Results showed that people with known benzene exposure (daily cigarette smokers) had higher levels of tt-MA and PMA compared with non-smokers. The method is advantageous for high-throughput analysis of selected VOC metabolites in large-scale, population-based studies such as the National Health and Nutrition Examination Survey (NHANES). Quantifying these urinary biomarkers is important to public health efforts to understand human exposure to VOCs from various sources, including tobacco products and electronic nicotine delivery systems.

Efficacy assessment of co-treated alpha-ketoglutarate and N-acetyl cysteine against the subchronic toxicity of cyanide in rats.

Cyanide is an important industrial pollutant, major occupational hazard, and a potential chemical warfare agent. Its intentional or accidental exposure to humans is a big clinical problem because of its rapid mode of action. Certain plant origin foods also contain substantial amount of cyanide and cause chronic toxicity. This study explores the protective efficacy of co-treatment of alpha-ketoglutarate (AKG) and an antioxidant N-acetyl cysteine (NAC) against toxicity of subchronically exposed cyanide in rats. We explore the effect of AKG + NAC co-treatment on oxidative stress, inflammation, and histological changes induced due to long-term sublethal cyanide exposure. Cyanide induces oxidative stress by inhibiting metalloenzymes (catalase and superoxide dismutase) causing increase in lipid peroxidation (malondialdehyde) and decrease in reduced glutathione (GSH). It also increases the activity of cyclo-oxygenase enzymes causing oxidative stress-mediated inflammation in the brain. Cyanide exposure also causes degenerative changes in the brain as shown in histology. It also causes pathology in liver and kidney. AKG is known to form cyanohydrins with cyanide reducing the free cyanide levels, and its combination with NAC showed overall improvement in by reducing the oxidative stress and subsequent neuroinflammation. Their combination was also found to improve the histological outcome of vital tissues. AKG, an over-the-counter sport medicine, and the antioxidant NAC per se did not show any detrimental effects in any tested parameter. Hence, oral treatment with AKG and NAC can be beneficial for the treatment of chronic cyanide poisoning.

Signaling by hydrogen sulfide and cyanide through post-translational modification.

Two cysteine metabolism-related molecules, hydrogen sulfide and hydrogen cyanide, which are considered toxic, have now been considered as signaling molecules. Hydrogen sulfide is produced in chloroplasts through the activity of sulfite reductase and in the cytosol and mitochondria by the action of sulfide-generating enzymes, and regulates/affects essential plant processes such as plant adaptation, development, photosynthesis, autophagy, and stomatal movement, where interplay with other signaling molecules occurs. The mechanism of action of sulfide, which modifies protein cysteine thiols to form persulfides, is related to its chemical features. This post-translational modification, called persulfidation, could play a protective role for thiols against oxidative damage. Hydrogen cyanide is produced during the biosynthesis of ethylene and camalexin in non-cyanogenic plants, and is detoxified by the action of sulfur-related enzymes. Cyanide functions include the breaking of seed dormancy, modifying the plant responses to biotic stress, and inhibition of root hair elongation. The mode of action of cyanide is under investigation, although it has recently been demonstrated to perform post-translational modification of protein cysteine thiols to form thiocyanate, a process called S-cyanylation. Therefore, the signaling roles of sulfide and most probably of cyanide are performed through the modification of specific cysteine residues, altering protein functions.

Deciphering the nitric oxide, cyanide and iron-mediated actions of sodium nitroprusside in cotyledons of salt stressed sunflower seedlings.

Nitric oxide (NO) is an endogenous signaling molecule in plants. Sodium nitroprusside (SNP), an established NO donor used in plant science research, simultaneously releases NO, cyanide (CN-) and iron (Fe) in solution. Since cyanide and iron mask NO effect of SNP, its use in NO research is debatable. Deciphering the action of SNP through NO, CN- or Fe has been undertaken in the present work. Cotyledons from salt stressed sunflower seedlings grown in the presence of NO donors were subjected to spectrofluorometric analysis of NO, CN- and Fe contents, and proteome and biochemical analyses. Diethylenetriamine NONOate (DETA) proved to be a better NO source since SNP enhanced ROS accumulation in the tissue. Abundance of 127 proteins is modulated by salt stress. SNP and exhausted SNP (exSNP) alter the abundance of 117 and 129 proteins, respectively. These proteins belong to primary metabolism, stress-response, transport, translation, proteolysis, chaperone, regulatory, and storage. Salt-responsive proteins, such as, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and isocitrate dehydrogenase are negatively modulated. DETA and SNP lower the activities of GAPDH and S-adenosylmethionine synthase (SAMS). Abundance of heat shock 70 kDa protein and actin are sensitive to both NaCl and NO. SNP affects plant growth by modulating proteome though iron, cyanide and NO. Its use only as an NO donor is thus debatable. exSNP control also releases substantial amount of cyanide and iron, thus questioning its use as control in NO research.

Metabolism of Cyanide by Glutathione To Produce the Novel Cyanide Metabolite 2-Aminothiazoline-4-oxoaminoethanoic Acid.

The direct analysis of cyanide (HCN or CN- inclusively symbolized as CN) to confirm exposure has major limitations due to cyanide's volatility, reactivity, and short half-life in biological fluids. These limitations have led to the exploration of cyanide detoxification products for indirect verification of cyanide exposure. Although cyanide interacts strongly with sulfur-containing molecules, to date, biomarkers resulting from the interaction of cyanide with glutathione (GSH; i.e., a biologically abundant sulfur-donating biomolecule) have yet to be discovered. In this study, we studied the interaction of CN and GSH to produce 2-aminothiazoline-4-oxoaminoethanioc acid (ATOEA). An LC-MS/MS method was developed and validated to analyze ATOEA from plasma, producing a linear range of 0.5-50 µM, a limit of detection of 200 nM, and excellent precision and accuracy. ATOEA concentrations were significantly elevated in the plasma of animals following cyanide exposure. Moreover, the production of ATOEA from cyanide exposure was confirmed by detection of both ATOEA and ATOEA-13C15N in rabbit plasma ( N = 11 animals) following administration of NaCN:K13C15N (1:1), with a similar amount of ATOEA and ATOEA-13C15N formed ( R2 = 0.9924, p < 0.05). The concentration of ATOEA increased with cyanide dose and then decreased rapidly when an antidote was administrated. This study definitively showed that ATOEA is produced from interaction of CN and GSH and can serve as a biomarker of cyanide exposure.