Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.

Formation of phenazines, phenoxazines, and benzoxazoles in the browning reactions of o-quinones.

Quinone-induced browning is widely produced in foods and is mostly considered a consequence of quinone/nucleophile reactions. However, even in the absence of amino acids or proteins, o-quinones develop browning. In an attempt to better understand the reaction pathways involved in this browning development, this study describes the reactions of 4-methyl-1,2-benzoquinone with alcohols, ammonia, and short chain aldehydes. These reaction mixtures developed browning at 37 °C and the main produced compounds were isolated by semipreparative HPLC and characterized by NMR and MS as phenazines, phenoxazines, and benzoxazoles. A reaction pathway that explains the formation of all these compounds is proposed. The formation of phenazines is responsible, at least partially, for the produced browning, and the formation of benzoxazoles inhibits such browning. Browning development seems to be a consequence of a competition among the reactions of formation of phenazines, phenoxazines, and benzoxazoles, which appear to be produced from a single intermediate.

Theanine in Tea: An Effective Scavenger of Single or Multiple Reactive Carbonyl Species at the Same Time.

Reactive carbonyl species (RCS) are generated during thermal food processing, and their accumulation in the body increases the risk of various chronic diseases. Herein, the RCS-scavenging ability of theanine, a unique nonproteinogenic amino acid, was evaluated in terms of the scavenging rate, reaction kinetics, and reaction pathway using LC-MS/MS. Three major products of theanine conjugated with acrolein (ACR) and glyoxal (GO) were prepared and identified using nuclear magnetic resonance. Thereafter, the simultaneous reactions of four types of RCS (namely, ACR, crotonaldehyde, methylglyoxal, and GO) with theanine were discussed in RCS-theanine and RCS-tea models. Under different reaction ratios, theanine could nonspecifically scavenge the four coexisting RCS by forming adducts with them. The amount of theanine-RCS adducts in green and black tea was more than that of catechin (epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin)-RCS adducts despite the lower content of theanine than catechins. Thus, theanine, as a food additive and dietary supplement, could demonstrate new bioactivity as a promising RCS scavenger in food processing.

The Hydrogenation of Crotonaldehyde on PdCu Single Atom Alloy Catalysts.

Recyclable PdCu single atom alloys supported on Al2O3 were applied to the selective hydrogenation of crotonaldehyde to elucidate the minimum number of Pd atoms required to facilitate the sustainable transformation of an α,ß-unsaturated carbonyl molecule. It was found that, by diluting the Pd content of the alloy, the reaction activity of Cu nanoparticles can be accelerated, enabling more time for the cascade conversion of butanal to butanol. In addition, a significant increase in the conversion rate was observed, compared to bulk Cu/Al2O3 and Pd/Al2O3 catalysts when normalising for Cu and Pd content, respectively. The reaction selectivity over the single atom alloy catalysts was found to be primarily controlled by the Cu host surface, mainly leading to the formation of butanal but at a significantly higher rate than the monometallic Cu catalyst. Low quantities of crotyl alcohol were observed over all Cu-based catalysts but not for the Pd monometallic catalyst, suggesting that it may be a transient species converted immediately to butanol and or isomerized to butanal. These results demonstrate that fine-tuning the dilution of PdCu single atom alloy catalysts can leverage the activity and selectivity enhancement, and lead to cost-effective, sustainable, and atom-efficient alternatives to monometallic catalysts.

Proteomics-based evaluation of the mechanism underlying vascular injury via DNA interstrand crosslinks, glutathione perturbation, mitogen-activated protein kinase, and Wnt and ErbB signaling pathways induced by crotonaldehyde.

Crotonaldehyde (CRA)-one of the major environmental pollutants from tobacco smoke and industrial pollution-is associated with vascular injury (VI). We used proteomics to systematically characterize the presently unclear molecular mechanism of VI and to identify new related targets or signaling pathways after exposure to CRA. Cell survival assays were used to assess DNA damage, whereas oxidative stress was determined using colorimetric assays and by quantitative fluorescence study; additionally, cyclooxygenase-2, mitogen-activated protein kinase pathways, Wnt3a, ß-catenin, phospho-ErbB2, and phospho-ErbB4 were assessed using ELISA. Proteins were quantitated via tandem mass tag-based liquid chromatography-mass spectrometry and bioinformatics analyses, and 34 differentially expressed proteins were confirmed using parallel reaction monitoring, which were defined as new indicators related to the mechanism underlying DNA damage; glutathione perturbation; mitogen-activated protein kinase; and the Wnt and ErbB signaling pathways in VI based on Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses. Parallel reaction monitoring confirmed significant (p < 0.05) upregulation (> 1.5-fold change) of 23 proteins and downregulation (< 0.667-fold change) of 11. The mechanisms of DNA interstrand crosslinks; glutathione perturbation; mitogen-activated protein kinase; cyclooxygenase-2; and the Wnt and ErbB signaling pathways may contribute to VI through their roles in DNA damage, oxidative stress, inflammation, vascular dysfunction, endothelial dysfunction, vascular remodeling, coagulation cascade, and the newly determined signaling pathways. Moreover, the Wnt and ErbB signaling pathways were identified as new disease pathways involved in VI. Taken together, the elucidated underlying mechanisms may help broaden existing understanding of the molecular mechanisms of VI induced by CRA.

Carbonyl-trapping abilities of 5-alkylresorcinols.

In an attempt to investigate the carbonyl-trapping abilities of 5-alkylresorcinols, this study describes the role of these compounds in inhibiting the formation of the 2,5-dialkylpyridines (5-ethyl-2-methylpyridine, 5-butyl-2-propylpyridine, and 5-hexyl-2-pentylpyridine) produced by 2-alkenals (crotonaldehyde, 2-hexenal, and 2-octenal) in the presence of ammonia. 5-Alkylresorcinols (as well as orcinol and olivetol) inhibited the formation of pyridines to an extend that depended on the 2-alkenal involved and the reaction conditions. This inhibition was consequence of the trapping of 2-alkenals by the phenolics. Thus, the major adducts produced between the C21:0 alkylresorcinol and crotonaldehyde were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). These results confirm that, in addition to their free radical scavenging abilities, 5-alkylresorcinols also trap reactive carbonyls. Because trapped carbonyls are involved in the formation of flavors and processing-induced antioxidants, 5-alkylresorcinols might be implied in some of the observed differences between whole and refined grain products.

Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

DABCO-based chiral ionic liquids as recoverable and reusable organocatalyst for asymmetric Diels-Alder reaction.

New DABCO-based chiral ionic liquids were synthesized and evaluated in asymmetric Diels-Alder reaction of cyclopentadiene with α,ß-unsaturated aldehydes or 4-phenyl-3-buten-2-one. Chiral ionic liquid of modified MacMillan catalyst having a DABCO cation and hexafluorophosphate anion acts as organocatalyst (5 mol%) for the Diels-Alder reaction of crotonaldehyde and cyclopentadiene producing 98% of the product and 87% ee (endo) in CH3 CN/H2 O (95/5) at 25°C in 2 h. The scope and limitations of the catalysis were also studied by using cyclopentadiene and α,ß-unsaturated aldehydes, and the Diels-Alder products were obtained in 18%-92% yields with 68%-93% ee. The catalyst was recycled and reused up to 6 cycles with a slight drop in ee and conversion of the product.

Inhibition of biological acidification and mechanism of crotonaldehyde removal with glucose cometabolism.

Biological acidification is an effective method in the treatment or pretreatment of industrial wastewater. Crotonaldehyde is a typical characteristic organic pollutant in petrochemical wastewater, but its effect on biological acidification is unclear. To investigate the inhibitory characteristic of crotonaldehyde on biological acidification and the mechanism of crotonaldehyde removal, variations in volatile fatty acid (VFA) yields, enzyme activities, biodegradation products, and microbial community structures were investigated by batch experiments in the presence of crotonaldehyde. The results showed that crotonaldehyde caused a 50% effect concentration (EC50) on the specific acidogenic activity (SAA) of 204.17 mg/L before 24 h, and then, the inhibitory effect was removed after 48 h as the dosage of crotonaldehyde was less than 1000 mg/L. Accordingly, crotonaldehyde was completely reduced to crotonyl alcohol by nicotinamide adenine dinucleotide (NADH) or oxidized to (E)-crotonic acid by aldehyde dehydrogenase (DHO) after 48 h. Next, 1-buanol and n-butyric acid were further metabolites, while the n-hexanoic acid detected with high-concentration crotonaldehyde might be due to the ORB pathway with 1-buanol as an electron donor. The dominant bacterial communities were Clostridium_sensu_stricto_1, Clostridium_sensu_stricto_11, Clostridium_sensu_stricto_12, which were related to the biodegradation process of crotonaldehyde. The findings of this research could provide a theoretical underpinning for developing the biological technologies to pretreat crotonaldehyde wastewater.

Predicting molecular mechanisms, pathways, and health outcomes induced by Juul e-cigarette aerosol chemicals using the Comparative Toxicogenomics Database.

There is a critical need to understand the health risks associated with vaping e-cigarettes, which has reached epidemic levels among teens. Juul is currently the most popular type of e-cigarette on the market. Using the Comparative Toxicogenomics Database (CTD; http://ctdbase.org), a public resource that integrates chemical, gene, phenotype and disease data, we aimed to analyze the potential molecular mechanisms of eight chemicals detected in the aerosols generated by heating Juul e-cigarette pods: nicotine, acetaldehyde, formaldehyde, free radicals, crotonaldehyde, acetone, pyruvaldehyde, and particulate matter. Curated content in CTD, including chemical-gene, chemical-phenotype, and chemical-disease interactions, as well as associated phenotypes and pathway enrichment, were analyzed to help identify potential molecular mechanisms and diseases associated with vaping. Nicotine shows the most direct disease associations of these chemicals, followed by particulate matter and formaldehyde. Together, these chemicals show a direct marker or mechanistic relationship with 400 unique diseases in CTD, particularly in the categories of cardiovascular diseases, nervous system diseases, respiratory tract diseases, cancers, and mental disorders. We chose three respiratory tract diseases to investigate further, and found that in addition to cellular processes of apoptosis and cell proliferation, prioritized phenotypes underlying Juul-associated respiratory tract disease outcomes include response to oxidative stress, inflammatory response, and several cell signaling pathways (p38MAPK, NIK/NFkappaB, calcium-mediated).

Atmospheric ozonolysis of crotonaldehyde in the absence and presence of hydroxylated silica oligomer cluster adsorption.

As one of the main components of combustion of tobacco products occurs (CARB), crotonaldehyde has an acute toxicity and widely exists in the atmosphere, which is harmful to human health. The removal efficiency of VOCs by ozonation can reach 80-90%. Based on the theory of quantum chemistry, the degradation mechanism, kinetics and toxicity of crotonaldehyde by ozonation in gas phase and heterogeneous phase were studied. Ozone was added to the olefins unsaturated double bond to form a five-membered ring primary ozonide, which was further fractured due to its unstable structure to form a Criegee intermediate and an aldehyde compound. The reaction rate constant of crotonaldehyde with ozone was 1.24 × 10-17 cm3 molecule-1 s-1 at 298 K and 1 atm, which was an order of magnitude higher than the experimental value. From toxicity assessment, it was found that the ozonation of crotonaldehyde is beneficial to the removal of toxicity. Mineral dust aerosol exists in the atmosphere in large quantities, and SiO2 is the most abundant component. VOCs are transformed into particle state on their surface through homogeneous nucleation and heterogeneous nucleation. Referring to the crystal structure of SiO2, five hydroxylated silica oligomer cluster structures were simulated and the adsorption configurations of crotonaldehyde on their surface were simulated. The adsorption of crotonaldehyde on the surface of the clusters was achieved by forming hydrogen bonds and had good adsorption effects. The adsorption of hydroxylated silica oligomer clusters didn't change the ozonation mechanism of crotonldehyde, but had a certain effect on the reaction rate.

[Determination of trace low molecular mass aldehydes in air by ultra-fast liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a rapid and accurate method for the determination of trace acetaldehyde, acrolein, methacrylaldehyde, crotonaldehyde in the air by ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS). METHODS: The air sample was concentrated and derivatived by 3-methyl-2(3 H)-benzothiazolonhydrazone(MBTH), and the effect of derivative conditions including the concentration of MBTH, the pH, the derivative time and temperature was investigated. The stability of derivatives, the cracking mechanism of mass spectrometry, the matrix effect of the method and air sampling efficiency were studied. The chromatographic separation was carried out on a Shim-pack XR-ODS II column(100 mm×2. 0 mm, 2. 2 µm) by using water/methanol solution as the mobile phase with the gradient elution. Detection was performed in positive multi-reaction monitoring(MRM) mode for quantification by using external standard method. RESULTS: The four analytes showed good linear relationship within the range of 1. 00-100 µg/L(calculated by aldehyde) with a correlation coefficient r≥0. 9990. The limits of quantitation(LOQs) of the method(concentrated with 10 L air) were 0. 5 µg/m~3 in air, for acetaldehyde, acrolein, methacrylaldehyde, crotonaldehyde. The recoveries of the method were 90. 6%-97. 8% at the three spiked levels, and the relative standard deviations(RSDs) were between 1. 9%-6. 4%(n=6), the average sampling efficiency was between 91. 0%-97. 1%. CONCLUSION: The developed method is simple, less interference and specificity, and is suitable for the simultaneous rapid determination of trace acetaldehyde, acrolein, methacrylaldehyde, crotonaldehyde in air of work place.

Crotonaldehyde exposure induces liver dysfunction and mitochondrial energy metabolism disorder in rats.

Crotonaldehyde is a highly toxic pollutant, widely present in tobacco smoke and automobile exhaust. Exposure to crotonaldehyde can cause hepatotoxicity and induce liver tumors in rats; however, the underlying mechanism is unclear. Liver cells contain many mitochondria, which serve to maintain energy levels in the body. We hypothesized that the energy metabolism disorder caused by mitochondrial dysfunction is an important cause of liver injury in rats exposed to crotonaldehyde. To test this, we randomly divided 40 male Wistar rats into four groups, and provided crotonaldehyde at 0, 2.5, 4.5, and 8.5 mg/kg for 90 days by intragastric administration. The results showed that crotonaldehyde exposure caused damage to liver mitochondrial structure, reduced electron-transport chain activity and ATP levels, and interfered with mitochondrial DNA transcription. In response to increased crotonaldehyde exposure, rats exhibited increased reactive oxygen species levels, decreased superoxide dismutase and glutathione activity, and activation of the caspase-mediated apoptosis pathway, as well as elevated levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, and histopathological damage. Our findings, together with those of previous reports, should help elucidate the underlying mechanism of crotonaldehyde-induced mitochondrial dysfunction and energy metabolism disorder, and provide an important direction for the prevention and clinical intervention of liver diseases caused by crotonaldehyde and aldehydes with similar structures.

Formation of naphthoquinones and anthraquinones by carbonyl-hydroquinone/benzoquinone reactions: A potential route for the origin of 9,10-anthraquinone in tea.

The reaction of 2-alkenals (crotonaldehyde and 2-pentenal) with hydroquinones (hydroquinone and tert-butylhydroquinone) and benzoquinones (benzoquinone, methylbenzoquinone, and methoxybenzoquinone) was studied as a potential route for the endogenous formation of naphthoquinones and anthraquinones in foods. Polycyclic quinones were produced at a low water activity, within a wide pH range, and in the presence of air. 9,10-Anthraquinone formation had an activation energy of 46.1 ± 0.1 kJ·mol-1, and a reaction pathway for the formation of the different naphthoquinones and anthraquinones is proposed. These reactions also took place in tea, therefore suggesting that the common tea pollutant 9,10-anthraquinone is also a process-induced contaminant. In fact, when four commercial teas (from a total of eight studied teas) were heated at 60 °C for 72 h, they significantly (p < 0.05) increased the amount of this toxicant. Reduction of 9,10-anthraquinone formation in teas is suggested to be carried out by reducing/scavenging its precursors.

Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach.

Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.

[A study of neurotoxicity of crotonaldehyde in male rats].

Objective: To investigate the neurotoxicity of crotonaldehyde exposure in male rats and its possible mechanism of action. Methods: From July to October 2019, 24 specific pathogen-free male Wistar rats were randomly divided into control group and 2.5, 4.5, and 8.5 mg/kg exposure groups, with 6 rats in each group, and the rats in these groups were given oral administration of crotonaldehyde solution at doses of 0.0, 2.5, 4.5, and 8.5 mg/kg, respectively, 5 times a week for 90 consecutive days. Body weight was measured after exposure, and brain tissue and liver tissue were collected. The activity of acetylcholinesterase (AChE) in brain tissue and the level of acetylcholine (ACh) in liver tissue were measured; The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the levels of malondialdehyde (MDA) and reduced glutathione (GSH) in brain tissue were measured; ELISA was used to measure the levels of interleukin-6 (IL-6) , interleukin-1ß (IL-1ß) , and tumor necrosis factor-α (TNF-α) in brain tissue. Results: Compared with the control group, the 2.5, 4.5, and 8.5 mg/kg exposure groups had a significant reduction in the activity of AChE in brain tissue, and the 8.5 mg/kg exposure group had a significant increase in the level of ACh in liver tissue (P<0.05) . Compared with the control group, the 4.5 and 8.5 mg/kg exposure groups had a significant increase in the level of MDA and significant reductions in the level of GSH and the activities of SOD and GSH-Px in brain tissue (P<0.05) . Compared with the control group, the 2.5, 4.5, and 8.5 mg/kg exposure groups had significant increases in the levels of TNF-α and IL-6 in brain tissue, and the 4.5 and 8.5 mg/kg exposure groups had a significant increase in the level of IL-1ß (P<0.05) . Conclusion: Crotonaldehyde exposure can induce nervous system injury in rats, possibly by altering oxidative balance and upregulating the expression of inflammatory factors in brain tissue.

Apoptosis of lung cells regulated by mitochondrial signal pathway in crotonaldehyde-induced lung injury.

Crotonaldehyde, a highly toxic α, ß-unsaturated aldehyde, is a ubiquitous hazardous pollutant. Because of its extreme toxicity and ubiquity in all types of smoke, most current research focuses on the lung toxicity of such air pollutants. However, the specific mechanism of pulmonary toxicity caused by crotonaldehyde remains unclear, especially after long-term exposure to crotonaldehyde at low dose. Therefore, the aim of the present study is to determine whether crotonaldehyde-induced oxidative damage and inflammation promote apoptosis in rats via the mitochondrial pathway using histopathology, immunohistochemistry, biochemistry analysis and Western blot analysis. The results show that crotonaldehyde elicited oxidative damage and inflammation in rats in a concentration-dependent manner. Crotonaldehyde-induced lung injury which was confirmed by H&E, Masson's trichrome staining and TUNEL. And crotonaldehyde-induced lung cell apoptosis showed a concentration-response relationship. Immunohistochemistry and Western blot results showed that apoptotic mitochondrial signaling pathway is abnormally activated in crotonaldehyde-induced lung injury. Collectively, this study demonstrates that exposure of rats to crotonaldehyde induces lung injury by inducing apoptosis, which is related to oxidative damage and inflammation through mitochondrial pathway.

Identification of Precursors and Formation Pathway for the Heterocyclic Aromatic Amine 2-Amino-3-methylimidazo(4,5-f)quinoline (IQ).

Food processing is responsible for the destruction of some health hazards, but it is responsible for the formation of new ones. Among them, the formation of heterocyclic aromatic amines (HAAs) has received a considerable attention because of their carcinogenicity. In spite of this, HAA formation is still poorly understood. This study was undertaken to identify precursors and formation pathways for 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). IQ was produced by reaction of acrolein, crotonaldehyde, creatinine, and ammonia. Reaction conditions were studied, and its activation energy (Ea) was determined to be 77.0 ± 1.3 kJ/mol. IQ formation was always accompanied by the formation of the HAA 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), which was produced with an Ea of 72.2 ± 0.4 kJ/mol. A reaction pathway for the competitive formation of IQ and MeIQ is proposed. Obtained results demonstrate the significant role of reactive carbonyls (the food carbonylome) in HAA formation and provide evidences for designing HAA mitigation strategies.

Reactive carbonyls and the formation of the heterocyclic aromatic amine 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ).

Reactions involving reactive carbonyls, creatinine, and ammonia-producing compounds were investigated in order to clarify the formation of the heterocyclic aromatic amine (HAA) 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ). Obtained results showed that MeIQ was only produced when 2-butenal (crotonaldehyde) was present. Reaction yields depended on the pH, with a maximum around pH 6.5, and on concentrations of crotonaldehyde and creatinine. Ammonia was also required for MeIQ formation, but ammonia was produced by creatinine decomposition. The amount of MeIQ formed increased with reaction time, temperature, and oxygen content in the reaction atmosphere. Activation energy for MeIQ formation from crotonaldehyde, creatinine, and glutamine was 72.2 ± 0.4 kJ·mol-1. A reaction pathway that explains MeIQ formation is proposed. Obtained results suggest a main role of reactive carbonyls formed in foods (the food carbonylome) on HAA formation. In addition, they provide scientific basis for the understanding of how HAAs are formed and could be mitigated.

Crotonaldehyde-induced vascular relaxation and toxicity: Role of endothelium and transient receptor potential ankyrin-1 (TRPA1).

INTRODUCTION: Crotonaldehyde (CR) is an electrophilic α,ß-unsaturated aldehyde present in foods and beverages and is a minor metabolite of 1,3-butadiene. CR is a product of incomplete combustion, and is at high levels in smoke of cigarettes and structural fires. Exposure to CR has been linked to cardiopulmonary toxicity and cardiovascular disease. OBJECTIVE: The purpose of this study was to examine the direct effects of CR in murine blood vessels (aorta and superior mesenteric artery, SMA) using an in vitro system. METHODS AND RESULTS: CR induced concentration-dependent (1-300 µM) relaxations (75-80%) in phenylephrine (PE) precontracted aorta and SMA. Because the SMA was 20× more sensitive to CR than aorta (SMA EC50 3.8 ± 0.5 µM; aorta EC50 76.0 ± 2.0 µM), mechanisms of CR relaxation were studied in SMA. The CR-induced relaxation at low concentrations (1-30 µM) was inhibited by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); 4) transient receptor potential ankyrin-1 (TRPA1) antagonist (A967079); and, 5) by non-vasoactive level of nicotine (1 µM). Similarly, a TRPA1 agonist, allyl isothiocyanate (AITC; mustard oil), stimulated SMA relaxation dependent on TRPA1, endothelium, NO, and GC. Consistent with these mechanisms, TRPA1 was present in the SMA endothelium. CR, at higher concentrations (100-300 µM), induced tension oscillations (spasms) and irreversibly impaired contractility (a vasotoxic effect enhanced by impaired endothelium). CONCLUSIONS: CR relaxation depends on a functional endothelium and TRPA1, whereas vasotoxicity is enhanced by endothelium dysfunction. Thus, CR is both vasoactive and vasotoxic along a concentration continuum.