Identification and apoptotic potential of T-2 toxin metabolites in human cells

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.     

Enzymatic synthesis and characterization of l-methionine and 2-hydroxy-4-(methylthio)butanoic acid (HMB) co-oligomers

Oligomers of l-methionine (Met) and its hydroxy analogue, 2-hydroxy-4-(methylthio)butanoic acid (d,l-HMB) were synthesized with the proteolytic enzyme papain. The Met homooligomers and HMB-Met co-oligomers obtained through the enzymatic reactions were subjected to persulfonation and separated with reverse phase liquid chromatography (RPLC). The separated oligomers were characterized with electrospray ionization-mass spectrometry (ESI-MS). The oligomers were also characterized with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that co-oligomers were predominantly composed of 4-8 Met residues and one HMB residue. The data also suggest that in the co-oligomers, HMB is attached at the N-terminal end of the oligopeptide chain.     

Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.     

Optimization of the synthesis of the cross-linked amino acid ornithinoalanine and nuclear magnetic resonance characterization of lysinoalanine and ornithinoalanine.

Lysinoalanine (LAL) and ornithinoalanine (OAL) are unnatural amino acids that can be formed in food submitted to thermal treatment, especially in alkaline conditions. The paper presents an optimization of the synthetic procedure for the preparation of a standard of OAL that could be very useful to study the toxicological and nutritional consequences of the presence of OAL in food. In the meantime, it was possible to develop a method based on nuclear magnetic resonance for the diastereomeric characterization of LAL and OAL without derivatization. Interest in this method is based on the known differences in the nephrotoxicity of the two diastereisomers of LAL.     

Detection and characterization of DNA adducts formed from metabolites of the fungicide ortho-phenylphenol

The significance of DNA adduction in ortho-phenylphenol-induced carcinogenesis remains unclear. Establishing adduct structures may contribute to resolving this issue. The chemical structures of the DNA adduction products resulting from the in vitro reaction of phenylbenzoquinone, the putative ultimate carcinogenic metabolite of the fungicide/disinfectant ortho-phenylphenol, are reported here. Three isomeric adducts that resulted from reaction of deoxyguanosine were characterized by UV, LC-ESI-MS, and MS/MS, and 1D and 2D COSY-NMR spectroscopy. The proposed mechanism of product formation is nucleophilic attack by the deoxyguanosine exocyclic amine nitrogen on an electrophilic quinone carbon, followed by stabilization through enolization. Another nucleophilic attack forms a five-membered ring, which aromatizes by dehydration to form the final product. Adducts were also characterized from deoxyadenosine and deoxycytidine, although conversions were at least 10 times lower. Structures are also proposed for these products. Cell culture studies confirmed that HepG2 cells incubated with phenylbenzoquinone at concentrations associated with cytotoxicity form the same DNA adducts.     

百菌清降解菌株H4的分离与表征及其修复污染土壤的潜能研究

A chlorothalonil(CTN)-degrading bacterial strain H4 was isolated in this study from a contaminated soil by continuous enrichment culture to identify its characteristics and to investigate its potential for remediation of CTN in contaminated soil. Based on the morphological, physiological and biochemical tests and 16 S r DNA sequence analysis, the strain was identified as Stenotrophomonas sp. After liquid culture for 7 d, 82.2% of CTN was removed by strain H4. The isolate could degrade CTN over a broad range of temperatures and p H values, and the optimum conditions for H4 degradation were p H 7.0 and 30℃. Reintroduction of the bacteria into artificially contaminated soil resulted in substantial removal of CTN( 50%) after incubation for 14 d. Soil samples treated by H4 showed significant increases(P 0.05) in soil dehydrogenase activity, soil polyphenol oxidase activity, average well-color development obtained by the Biolog Eco plate TM assay and Shannon-Weaver index, compared with the control. Strain H4 might be a promising candidate for application in the bioremediation of CTN-contaminated soils.     

Kinetic characterization of the enzymatic and chemical oxidation of the catechins in green tea

The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.     

In vitro stability and chemical reactivity of thiosulfinates

A model reaction system was used to generate pure thiosulfinates (3) from S-alk(en)yl-L-cysteine sulfoxides (1) to facilitate studies on the intrinsic pH and thermal sensitivities of individual thiosulfinate species. Thiosulfinate decay could be characterized as first-order processes over the pH range of 1.2-9.0 and at 20-80 degrees C. The stability of thiosulfinates was greatest at pH 4.5-5.5, followed by pH 1.2, pH 6.5-7.5, and pH 8.0-9.0. Thiosulfinates with longer and saturated alk(en)yl groups were generally more stable than those with shorter and unsaturated alk(en)yl groups. Thiosulfinates underwent thioalkyl-exchange reactions at pH 8-9 without loss of total thiosulfinate levels within 60-90 min at 20 degrees C.     

Biological and chemical characterization of harbour sediments from the Stockholm area

Purpose  

The main objective of the current study was to assess the impact of pleasure boat activities on harbour sediment quality in the Stockholm area. Sediment contamination is a growing ecological issue, and there is consequently a need to use sediment bioassays in combination with chemical analysis to determine the impact on the ecosystem. To generate sediment toxicity data relevant for the Baltic Sea, a secondary objective was to further develop and evaluate two well-established bioassays for saltwater, with the macroalga Ceramium tenuicorne and the crustacean Nitocra spinipes, to be useful also for toxicity testing of whole sediment. A major concern has been to minimize any manipulation of the sediments. A third objective was to assess whether a simple leaching procedure could be used to simulate sediment toxicity by comparing results from whole sediment and leachate tests.     

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3'-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.     

Antioxidant activity of isoflavones and their major metabolites using different in vitro assays

Isoflavone phytoestrogens found mainly in soybeans and clover are widely studied phytochemicals. Genistein and daidzein, the major isoflavones found in soy, have received the most attention. However, they undergo extensive metabolism in the intestine and the liver, which might affect their biological properties, e.g. their antioxidant capacities. Furthermore, the biological activities of other naturally occurring isoflavones, for instance, glycitein from soy or biochanin A from red clover, have not yet been studied in detail. The aim of this study was to investigate the antioxidant activities of six naturally occurring isoflavones and their corresponding oxidative and bacterial metabolites. The oxygen radical absorbance capacity assay as well as the in vitro oxidation of low density lipoproteins with the conjugated diene and the thiobarbituric acid reacting substances formation as end points were used. The oxidative metabolites of genistein and daidzein as well as equol exhibited the highest antioxidant activities in all three assays. With few exceptions, they were more effective than the positive controls quercetin and ascorbic acid. Formononetin, the 4'-O-methyl ether of daidzein, showed the lowest antioxidant property. Because the antioxidant efficacy of isoflavones as effective antioxidants is evident at concentrations well within the range found in the plasma of subjects consuming soy products, this biological activity could be of physiological relevance.     

Identification of 4-O-5'-coupled diferulic acid from insoluble cereal fiber

The extracts of saponified cereal fibers of whole grains of corn (Zea mays cv. microsperma KOERN.), wheat (Triticum aestivum L.), spelt (Triticum spelta L.), and rice (Oryza sativa L.) were investigated for dehydrodimers of ferulic acid using gas-liquid chromatography (GLC) with mass spectrometric detection (GLC-MS) and flame ionization detection (GLC-FID). In addition to the 8,5'-, 8, 8'-, 5,5'-, and 8-O-4'-coupled diferulic acids previously identified from other plant materials the 4-O-5'-coupled diferulic acid (E)-3-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-5-methoxyci nnamic acid (4-O-5'-DFA) was identified in all fibers investigated. This new diferulate was authenticated by comparison of its mass spectrum and its relative GLC retention time with those of the synthesized compound. Semiquantitative determination of 4-O-5'-DFA showed that it is present at 8-30 microg/g, approximately 70-100 times lower concentrations than the sum of 8,5'-coupled diferulic acids, the major diferulic acids in the investigated fibers.     

Method for analysis of tannic acid and its metabolites in biological samples: application to tannic acid metabolism in the rat

A new analytical method for measuring tannic acid (TA) using tannase was developed and applied to the investigation of TA metabolism in the rat following oral administration at a dose of 1.0 g/kg. The proposed method for TA determination was based on the enzymatic hydrolysis of TA to gallic acid (GA) and subsequent determination by HPLC. TA metabolites were determined by HPLC. 4-O-Methylgallic acid (4-OMGA), pyrogallol (PY), and resorcinol (RE) were detected in serum. TA was excreted into urine as GA (0.01%), 4-OMGA (0.10%), PY (0.24%), and RE (2.06%) and into feces as TA (62.74%), GA (0.19%), PY (0.02%), and RE (0.76%) within 54 h after oral administration. It was suggested that >60% of TA remained unchanged but that some was hydrolyzed to GA by tannase in the intestine and further metabolized to 4-OMGA, PY, and RE.     

Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.     

Studies of the in vitro intestinal metabolism of isoflavones aid in the identification of their urinary metabolites

Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and alpha-methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.     

In vitro inhibition of human cGMP-specific phosphodiesterase-5 by polyphenols from red grapes

A moderate consumption of red wine may reduce the risk of cardiovascular diseases via wine-derived phenolic compounds. A variety of biological mechanisms have been proposed for wine-derived phenolic compounds including nitric oxide-mediated vasorelaxation. This study examined whether the vasodilating effect of wine-derived phenolic compounds was associated with the inhibition of phosphodiesterases (PDEs) and, in particular, PDE5. For this purpose, human recombinant PDE5A1 isoform was prepared by expression of the full-length cDNA of PDE5A1 into COS-7 cells. Red wine and the extracts from grape skin inhibited PDE5A1 activity, whereas the seed extracts had a negligible effect. The mixture of anthocyanins inhibited the enzyme activity (IC50 = 11.6 microM), with malvidin-3-O-beta-glucoside (IC50 = 35.4 microM) and malvidin (IC50 = 24.9 microM) the most potent among the monoglucosides and aglycons, respectively. trans-Resveratrol and trans-piceid exhibited negligible effects, whereas hydroxycinnamates were completely inactive. These results indicate that polyphenols-induced vasorelaxation may also be sustained by smooth muscle PDE inhibition by anthocyanins present in red wines and grapes.     

Synthesis and characterization of N-acylaniline derivatives as potential chemical hybridizing agents (CHAs) for wheat (Triticum aestivum L.)

Induction of male sterility by deployment of chemical hybridizing agents (CHAs) are important in heterosis breeding of self-pollinated crops like wheat, wherein the male and female organs are in the same flower. Taking a lead from the earlier work on rice, a total of 25 N-acylanilines comprising of malonanilates, acetoacetanilides, and acetanilides (including halogenated acetanilides) were synthesized and screened as CHAs on three genotypes of wheat, viz., PBW 343, HD 2046, and HD 2733 at 1500 ppm in the winter of 2001-2002. The N-acylanilines containing variations at the acyl and aromatic domain were synthesized by condensation of substituted anilines with appropriate diesters, acid chlorides, or monoesters. The test compounds with highly electronegative groups such as F/Br at the para position of the aryl ring were identified as the most potent CHAs, causing higher induction of male sterility. A variation of N-substitution at the side chain generally furnished analogues like 4'-fluoroacetoacetanilide (7) and ethyl 4'-fluoromalonanilate (1), which induced 89.12 and 84.66% male sterility, respectively, in PBW 343. Among halogenated acetanilides, the increasing number of chlorine atoms in the side chain led to an increase in the activity of 4'-fluoro (23) and 4'-bromo (24) derivatives of trichoroacetanilides, which induced >87% male sterility. Quantitative structure-activity relationship (QSAR) models indicated the positive contributions of the field effect exemplified by the Swain-Lupton constant (Fp) and negative contributions of the Swain-Lupton resonance constant (R) for the aromatic substitution. The positive influences of parachor (P) for the acyl domain have been underlined. These leads will be significant in explaining the CHA fit in the macromolecular receptor site. The CHAs appeared to act by causing an imbalance in the acid-base equilibrium in pollen mother cells resulting in dissolution of the callose wall by premature callase secretion.     

Rivrisk: A model to assess potential human health and ecological risks from chemical and thermal releases into rivers

RIVRISK, an interactive WindoWSTM-based model, predicts ambient chemical concentrations, human health and ecological risks, and water temperatures due to chemical and thermal releases into rivers. RIVRISK simulates chemicals that enter rivers from pipes, through submerged diffusers, from groundwater seepage, and atmospheric plume deposition. Human health risks are calculated for up to 16 pathways via exposure routes of dermal contact, ingestion, and inhalation. Ecological risks are evaluated for aquatic organisms by comparing the river water concentration to a water quality criterion for freshwater organisms. Monte Carlo analyses of ambient concentrations and temperatures can also be performed based on uniform distributions or user-defined histograms of varied parameters. To illustrate the various features of the model, RIVRISK was applied to a case study based on the Glen Lyn Power Plant located along the New River in Virginia.     

保水剂对干旱胁迫下雪茄烟叶次生代谢物质及化学成分含量的影响

为研究干旱胁迫下保水剂种类及用量对雪茄烟叶次生代谢物质及化学成分含量的影响,以德雪3 号为试材,采用盆栽试验研究了保水剂种类(SAP保水剂、沃特保水剂和自制TS-PAA保水剂)与用量(2、4和6 g·株-1)对雪茄烟生长过程中质体色素、多酚、有机酸含量的消长规律及其调制后化学成分含量变化的影响。结果表明,①干旱胁迫下施用3 种保水剂均能显著增加烟叶中叶绿素、叶绿素a、叶绿素b和类胡萝卜素的含量,且有利于烟株前中期叶绿素的快速积累和后期叶绿素的充分降解,以确保烟株适时落黄;②生长发育期间绿原酸、芸香苷、莨菪亭含量呈先增加后缓慢降低的趋势,且显著高于对照,说明施加3 种保水剂均有利于多酚类物质的积累,能间接提升调制后烟叶的香吃味;③在移栽后75 d时,3 种保水剂处理下苹果酸、草酸、丙二酸含量均有不同程度的提高,而柠檬酸含量与对照无显著差异;④施加3 种保水剂均能显著提高总糖、还原糖和钾含量,并降低总氮、烟碱、淀粉和氯含量,进而增加两糖比、糖碱比、钾氯比和氮碱比,使烟叶中化学成分更加均衡协调。不同处理相比较,TS-PAA保水剂和SAP保水剂以施用量为4 g·株-1,沃特保水剂以施用量为6 g·株-1时对次生代谢物质积累和烟叶质量改善作用效果最佳。