2-hexenal--z-isomer and 2-butenal

A highly sensitive, selective and reproducible chromatographic method is described for determination of low-molecular mass unsaturated aliphatic aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivatives were separated on a reversed-phase column C8 isocratically using a mixture of acetonitrile and 90mM imidazole-HNO3 buffer (pH 6.4, 1:1, % v/v). The calibration ranges were: 20-420nM for methylglyoxal, 16-320nM for acrolein, 15-360nM for crotonaldehyde and 20-320nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5nM (88-130fmol/injection), the recovery results were within the range of 87.4-103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients' sera with simple pretreatment method. In conclusion, this new method is suitable for routine analysis of large numbers of clinical samples for assessment of the oxidative stress state in patients.

Topics: Adult; Aged; Aged, 80 and over; Aldehydes; Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Diabetes Mellitus; Female; Fluorescent Dyes; Humans; Lipid Peroxidation; Male; Middle Aged; Molecular Weight; Oxadiazoles; Oxidative Stress; Reproducibility of Results; Spectrometry, Fluorescence; Sulfonamides

The effects of 16 aliphatic aldehydes with 3-10 carbons on the growth and patulin production of Penicillium expansum were examined. When P. expansum spores were inoculated into apple juice broth, some alkenals, including 2-propenal, (E)-2-butenal, (E)-2-pentenal, and (E)-2-hexenal, inhibited fungal growth and patulin production. Their minimal inhibitory concentrations were 5, 50, 80, and 80 µg/mL respectively. Vital staining indicated that these alkenals killed mycelia within 4 h. Treatment of the spores with these aldehydes also resulted in rapid loss of germination ability, within 0.5-2 d. On the other hand, aliphatic aldehydes with 8-10 carbons significantly enhanced patulin production without affecting fungal growth: 300 µg/mL of octanal and 100 µg/mL of (E)-2-octenal increased the patulin concentrations in the culture broth by as much as 8.6- and 7.8-fold as compared to that of the control culture respectively. The expression of the genes involved in patulin biosynthesis in P. expansum was investigated in mycelia cultured in apple juice broth containing 300 µg/mL of octanal for 3.5, 5, and 7 d. Transcription of the msas gene, encoding 6-methylsalicylic acid synthase, which catalyzed the first step in the patulin biosynthetic pathway was remarkably high in the 3.5-d and 5-d-old cultures as compared with the control. However, octanal did not any increase the transcription of the msas in the 7-d-old culture or that of the other two genes, IDH and the peab1, in culture. Thus the enhanced patulin accumulation with supplementation with these aldehydes is attributable to the increased amount of the msas transcript.

Topics: Acrolein; Acyltransferases; Aldehydes; Beverages; Fermentation; Fruit; Fungal Proteins; Gene Expression; Ligases; Malus; Multienzyme Complexes; Mycelium; Oxidoreductases; Patulin; Penicillium; Spores, Fungal; Transcription, Genetic

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO(2) photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. alpha,beta-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO(2)-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H(2)O --> ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.

Topics: Acrolein; Aldehydes; Ascorbic Acid; Chloroplasts; Cysteine; Electron Transport; Enzyme Activation; Glutathione; Light; Oxidation-Reduction; Photosynthesis; Spinacia oleracea; Stress, Physiological

Induction of pumpkin (Cucurbita maxima Duch.) glutathione S-transferase (GST, EC 2.5.1.18) by aldehydes and related compounds was examined. All of the tested compounds induced pumpkin GST to different degrees, and it was found that (1) aldehydes induce GST directly and alcohols induce GST indirectly, and (2) alpha,beta-unsaturated aldehydes are the most effective inducers and their potency is related to the Michael acceptors reaction. The results of Western blot analysis showed that the patterns of induction of CmGSTU1, CmGSTU2 and CmGSTU3 were similar to the patterns of activity with the exception of alpha,beta-unsaturated carbonyl compounds. Among the three compounds, crotonaldehyde caused the highest activity induction (9.2-fold), but Western blot expression was the highest only for CmGSTU3. CmGSTF1 was almost non-responsive to all of the tested stresses. Results of induction studies suggested that efficient pumpkin GST inducers have distinctive chemical features. The in vitro activity of the enzyme was inhibited by ethacryanic acid, trans-2-hexenal, crotonaldehyde, and pentanal. Ethacryanic acid was found to be the most potent inhibitor with an apparent I(50) value of 6.90+/-2.06 micro M, while others were weak to moderate inhibitors. The results presented here indicate that plant GSTs might be involved in the detoxification of physiologically and environmentally hazardous aldehydes/alcohols.

Topics: Alcohols; Aldehydes; Blotting, Western; Cucurbita; Enzyme Induction; Ethacrynic Acid; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glutathione Transferase

Lipid peroxidation due to oxidative stress is accelerated under hyperglycemic conditions such as diabetes mellitus. The effect of 4-hydroxy-2-nonenal (HNE) and other lipid peroxidation products on the ability of isolated rat pancreatic islets to secrete insulin was examined in this study. HNE concentration- and time-dependently deteriorated glucose-induced insulin secretion: insulin secretion was decreased by 50% when measured after incubation of islets with 100 microM HNE for 1 h. Other lipid peroxidation products, e.g. 2-hexenal and 2-butenal, also inhibited glucose-induced insulin secretion. HNE at 100 microM lowered alpha-ketoisocaproate-induced insulin secretion, whereas leucine-induced insulin secretion was stimulated. Insulin secretion induced by 10 mM glyceraldehyde was slightly decreased by HNE. On the other hand, HNE severely decreased insulin secretion induced by 10 mM glyceraldehyde and 2.8 mM glucose. Glucose utilization and glucose oxidation were significantly lowered in islets treated with HNE. The amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate in islets were decreased by treatment with HNE, whereas the amount of fructose 6-phosphate was increased. Our study indicates that HNE and other lipid peroxidation products impair insulin secretion induced by glucose probably through affecting both the glycolytic pathway and the citric acid cycle.

Topics: Aldehydes; Animals; Citric Acid Cycle; Dihydroxyacetone Phosphate; Female; Fructosediphosphates; Glucose; Glyceraldehyde; Glycolysis; Insulin; Insulin Secretion; Islets of Langerhans; Keto Acids; Leucine; Lipid Peroxidation; Oxidative Stress; Rats; Rats, Wistar

Crotonaldehyde and 2-hexenal are bifunctional compounds that form 1,N2-propanodeoxyguanosine adducts and are mutagenic and genotoxic; crotonaldehyde is carcinogenic. Analysis of the mutations resulting from crotonaldehyde-induced DNA damage revealed the importance of deoxyguanosine adducts. Humans are exposed ubiquitously to these compounds by various routes. The highest daily intake of crotonaldehyde is assumed to be derived from cigarette smoke (31-169 micrograms/kg body weight), and the highest intake of 2-hexenal is probably from fruit and vegetables (31-165 micrograms/kg body weight per day). Because these compounds are suspected to play on important role in carcinogenicity, we developed sensitive 32P-postlabelling techniques for DNA adducts of crotonaldehyde and hexenal, in order to improve estimates of cancer risk. The respective standards were also synthesized and characterized spectroscopically. We report here the results of the 32P-postlabelling, e.g. the stability of the adducts in respect of nuclease P1 treatment, their labelling efficiencies, thin-layer chromatography of adduct spots and the recoveries and detection limits. In untreated male Fischer 344 rats, neither crotonaldehyde nor 2-hexenal adducts were detected, but crotonaldehyde adducts were found in the tissues of rats given single doses of 200 or 300 mg/kg body weight and in the livers of rats after repeated doses of 1 or 10 mg/kg body weight. The adduct levels were higher 20 h after gavage than after 12 h. The adducts persist to a certain extent. 2-Hexenal adducts were detected in tissues of male Fischer 344 rats after gavage with single doses of 50, 200 or 500 mg/kg body weight. The highest adduct levels were measured 48 h after gavage, but no adducts were found 8 h after gavage. Two approaches for cancer risk estimation are discussed. One is based on the correlation between the covalent binding index, calculated from adduct levels, and the median toxic dose (TD50) (Lutz, 1986) and showed a cancer risk of 1 per 10(7) lives for hexenal, assuming dietary intakes of 31-165 micrograms/kg body weight per day. The other is based on a cancer incidence of 0.07 at a dose of crotonaldehyde of 4.2 mg/kg body weight per day assessed from the study of Chung et al. (1986), which can be interpreted as a risk of 5.8-18 new cases per 10(4) smokers, assuming a consumption of 30 cigarettes per day. The latter approach may, however, lead to an overestimate of the cancer risk associated with exposure

Topics: Adenosine Triphosphate; Aldehydes; Animals; Carcinogens; Chromatography, Thin Layer; Deoxyguanosine; Diet; DNA Adducts; DNA Damage; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Kinetics; Liver; Male; Models, Chemical; Mutagens; Neoplasms; Polynucleotide 5'-Hydroxyl-Kinase; Rats; Rats, Inbred F344; Risk Assessment; Tissue Distribution

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.

Topics: Aldehydes; Chromatography, Liquid; Deoxyadenosines; Deoxyguanosine; DNA Adducts; Lipid Peroxidation; Malondialdehyde; Mass Spectrometry; Reproducibility of Results; Sensitivity and Specificity

In the present study the irreversible inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by alpha, beta-unsaturated aldehydes and ketones was studied. When GSTP1-1 was incubated with a 50-fold molar excess of the aldehydes acrolein (ACR) and 4-hydroxy-2-nonenal (HNE) and the ketones curcumin (CUR) and ethacrynic acid (EA) at 22 degrees C, all of them inactivated GSTP1-1. The remaining activity after 4 h of incubation in all cases was lower than 10%. The aldehydes crotonaldehyde (CRA), cinnamaldehyde (CA) and trans-2-hexenal were found to inhibit GSTP1-1 only at a 5000-fold molar excess and even then, for example, for CA a higher remaining activity of 17% was observed. The same inhibition experiments were conducted with 3 mutants of GSTP1-1: the C47S and C101S mutants and the double mutant C47S/C101S. Remaining activity for C47S varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. For C101S it varied between 0 and 9% and for the double mutant C47S/C101S, activity after 4 h of incubation was variable. Again it varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. EA is known to react almost exclusively with cysteine 47. When [14C]EA was incubated with the GSTP1-1, modified by the alpha, beta-unsaturated carbonyl compounds, no [14C]EA was incorporated in the enzyme, indicating that in all cases this cysteine residue was one of the major targets. Since Michael addition with these reagents is known to be reversible, the results of incubation of the inactivated enzymes with an excess of glutathione (GSH) were determined. For all compounds, a restoration of the catalytic activity was observed. The results indicate that alpha, beta-unsaturated carbonyl derivatives inhibit GSTP1-1 irreversibly mainly by binding to cysteine residues of GSTP1-1, especially Cys-47, This means that some of these compounds (e.g. CUR) might modify GST activity in vivo when GSH concentrations are low by covalent binding to the enzyme.

Topics: Acrolein; Aldehydes; Curcumin; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Ethacrynic Acid; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Ketones; Mutagenesis, Site-Directed

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.

Topics: Acrolein; Acyclic Monoterpenes; Aldehydes; Chromatography, High Pressure Liquid; Curcumin; Enzyme Inhibitors; Ethacrynic Acid; Glutathione; Glutathione Transferase; Humans; Melanoma; Monoterpenes; Skin Neoplasms; Terpenes; Tumor Cells, Cultured