Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries

A total of 94 European eels (Anguilla anguilla) were collected from five estuaries in the UK. The deconjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the eels were separated using HPLC. Six PAH metabolites were identified: 1-hydroxy (1-OH) metabolites of phenanthrene, pyrene and chrysene; and the 1-OH, 3-OH and 7,8 dihydrodiol metabolites of benzo[a]pyrene (BaP). The mean concentration of the six metabolites was greatest in eels from the Tyne (49 muM) followed by the Wear (33 muM), Tees (19 muM), Thames (4 muM) and Severn (2 muM) estuaries. Although 1-OH pyrene was always the dominant compound, there were significant differences (P<0.05) between sites and between estuaries for some metabolites. Normalising the molar concentration of the bile metabolites to the bile biliverdin absorbance reduced sample variation. When the metabolites identified were-each expressed as a percentage of the total detected, the metabolite profile was characteristic for each estuary. (C) 2002 Elsevier Science B.V. All rights reserved.

Simple and surprisingly effective one-step extraction-cleanup by Soxflo for DDT and its metabolites from environmental samples

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some-but not all-samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 mug/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC. (C) 2004 Elsevier B.V. All rights reserved.

Microbial control of diamondback moth, Plutella xylostella L. (Lepidoptera:Yponomeutidae) using bacteria (Xenorhabdus nematophila) and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X. nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

Biological control of black vine weevil (Otiorhynchus sulcatus, Coleoptera: Curculionidae) by entomopathogenic bacteria and their cell-free toxic metabolites

Biocontrol agents such as Xeiwrhabduf, nemalophilci and X. nematophila ssp. bovienii and their cell-free protein toxin complexes were lethal to larvae of O. sulcatus when applied to potting compost in the absence of plants. Similarly, strawberry plants infected with 0. sulcaitfi larvae were protected from damage by applications of both cell suspensions of the bacteria and solutions of their cell-free toxic metabolites, indicating that it is the protein toxins, which are responsible for the lethal effects observed. These toxic metabolites were found more effective against 0. sulccitus larvae when treated in soil microflora. Insect mortality is increased by increasing temperature and bacterial concentration. The toxins remained pathogenic for several months when stored in potting soil either at 15 or 20°C, however, bacterial cells were not as persistent as the toxins. It is therefore suggested that these bacteria and their toxic metabolites can he applied in soil for insect pest control.

An evaluation of the costs of making specific secondary metabolites: does the yield penalty incurred by host plant resistance to insects due to competition for resources?

The presumption that the synthesis of 'defence' compounds in plants must incur some 'trade-off' or penalty in terms of annual crop yields has been used to explain observed inverse correlations between resistance to herbivores and rates of growth or photosynthesis. An analysis of the cost of making secondary compounds suggests that this accounts for only a small part of the overall carbon budget of annual crop plants. Even the highest reported amounts of secondary metabolites found in different crop species (flavonoids, allylisothiocyanates, hydroxamic acids, 2-tridecanone) represent a carbon demand that can be satisfied by less than an hour's photosynthesis. Similar considerations apply to secondary compounds containing nitrogen or sulphur, which are unlikely to represent a major investment compared to the cost of making proteins, the major demand for these elements. Decreases in growth and photosynthesis in response to stress are more likely the result of programmed down-regulation. Observed correlations between yield and low contents of unpalatable or toxic compounds may be the result of parallel selection during the refinement of crop species by humans.

In vitro antioxidant activity of coffee compounds and their metabolites

In this paper we report the antioxidant activity of different compounds which are present in coffee or are produced as a result of the metabolism of this beverage. In vitro methods such as the ABTS(center dot+) [ABTS = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay and the oxygen radical absorbance capacity assay (ORAC) were used to assess the capacity of coffee compounds to scavenge free radicals. The importance of caffeine metabolites and colonic metabolites in the overall antioxidant activity associated with coffee consumption is shown. Colonic metabolites such as m-coumaric acid and dihydroferulic acid showed high antioxidant activity. The ability of these compounds to protect human low-density lipoprotein (LDL) oxidation by copper and 2,2'-azobis(2-amidinopropane) dihydrochloride was also explored. 1-Methyluric acid was particularly effective at inhibiting LDL oxidative modification. Different experiments showed that this caffeine metabolite is not incorporated into LDL particles. However, at physiologically relevant concentrations, it was able to delay for more than 13 h LDL oxidation by copper.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Antioxidant and free radical scavenging activity of isoflavone metabolites

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

A new route to furanoeremophilane sesquiterpenoids. Synthesis of Senecio metabolites (+/-)-6-hydroxyeuryopsin, (+/-)-1,10-epoxy-6-hydroxyeuryopsin, (+/-)-toluccanolide A and (+/-)-toluccanolide C

A new strategy for the synthesis of sesquiterpenoids of the furanoeremophilane family was developed in which the tricyclic nucleus was assembled in an A + C -> A - C -> A - B - C sequence. The A - C connection was made via coupling of a cyclohexenylmethyl bromide with a stannylfuran under "ligandless" Stille conditions, and the key cyclization which closed ring B was accomplished with complete stereocontrol by intramolecular formylation of a 2-silylfuran in the presence of trimethylsilyl triflate. This route was used to complete the first total syntheses of the furanoeremophilane 6-hydroxyeuryopsin and the eremophilenolides toluccanolide A and toluccanolide C, as well as a formal synthesis of 1,10-epoxy-6-hydroxyeuryopsin.

Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity

Flavonoids have been proposed to act as beneficial agents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. The biological effect of these polyphenols and their in vivo circulating metabolites will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or more importantly their uptake. This review summarises the current knowledge on the cellular uptake of flavonoids and their metabolites with particular relevance to further intracellular metabolism and the generation of potential new bioactive forms. Uptake and metabolism of the circulating forms of flavanols, flavonols, and flavanones into cells of the skin, the brain, and cancer cells is reviewed and potential biological relevance to intracellular formed metabolites is discussed.

Antioxidant and free radical scavenging activity of isoflavone metabolites

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono-and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity. (c) 2006 Elsevier Inc. All rights reserved.

Effect of colonic bacterial metabolites on Caco-2 cell paracellular permeability in vitro

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [C-14]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.

Absorption, tissue distribution and excretion of pelargonidin and its metabolites following oral administration to rats

Recent reports have demonstrated various cardiovascular and neurological benefits associated with the consumption of foods rich in anthocyanidins. However, information regarding absorption, metabolism, and especially, tissue distribution are only beginning to accumulate. In the present study, we investigated the occurrence and the kinetics of various circulating pelargonidin metabolites, and we aimed at providing initial information with regard to tissue distribution. Based on HPLC and LC-MS analyses we demonstrate that pelargonidin is absorbed and present in plasma following oral gavage to rats. In addition, the main structurally related pelargonidin metabolite identified in plasma and urine was pelargonidin glucuronide. Furthermore, p-hydroxybenzoic acid, a ring fission product of pelargonidin, was detected in plasma and urine samples obtained at 2 and 18 h after ingestion. At 2 h post-gavage, pelargonidin glucuronide was the major metabolite detected in kidney and liver, with levels reaching 0.5 and 0.15 nmol pelargonidin equivalents/g tissue, respectively. Brain and lung tissues contained detectable levels of the aglycone, with the glucuronide also present in the lungs. Other tissues, including spleen and heart, did not contain detectable levels of pelargonidin or ensuing metabolites. At 18 h post-gavage, tissue analyses did not reveal detectable levels of the aglycone nor of pelargonidin glucuronides. Taken together, our results demonstrate that the overall uptake of the administered pelargonidin was 18 % after 2 h, with the majority of the detected levels located in the stomach. However, the amounts recovered dropped to 1.2 % only 18 h post-gavage, with the urine and faecal content constituting almost 90 % of the total recovered pelargonidin.