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Evaluation of the allergic/irritant potential of air pollutants: detection of proteins modified by volatile organic compounds from oilseed rape (Brassica napus ssp. oleifera) using electrospray ionization-mass spectrometry.

BACKGROUND: Upward trends in allergy and asthma rates have been reported in most western societies, including the UK, where around 15-20% of the population now suffer from allergy or asthma. Scientific proof of the causes of these increases relies on accurate assessment of exposure and standardized diagnostic tests, such as for specific IgE in blood serum and skin testing. For many air pollutants it has proven difficult to assess an individual's exposure outside an occupational environment and reliable test development is hampered by not knowing whether an allergic or irritant mechanism is involved. These problems are particularly evident in the controversial issue of whether airborne releases from oilseed rape can cause health effects. OBJECTIVE: To develop a method for evaluating the allergic/irritant potential of air pollutants and to assess whether the volatile organic compounds emitted by oilseed rape have this potential. METHODS: Proteins were exposed in vitro to volatile organic compounds emitted by oilseed rape. Electrospray ionization-mass spectrometry was used to detect any resultant protein modifications. RESULTS: Dimethyl disulphide, thiocyanic acid methyl ester and 2-methyl-propanenitrile were able to modify human proteins. In addition, two isothiocyanates which can be emitted by damaged oilseed rape also have this ability. The major products emitted by undamaged oilseed rape, terpenes, a sesquiterpene and a terpene alcohol did not have this property, but the possible role of their oxidized products is discussed. CONCLUSION: Some of the volatile organic compounds emitted by oilseed rape have the potential to be allergens/irritants. Standardized modified proteins produced by this method should prove useful for biomonitoring human exposure in molecular epidemiological studies as well as in diagnostic tests. This method should find further application in investigations into the possible health effects of other environmental pollutants.

Air Pollutants↗

Asthmatic symptoms and volatile organic compounds, formaldehyde, and carbon dioxide in dwellings.

OBJECTIVES: As a part of the worldwide European Community respiratory health survey, possible relations between symptoms of asthma, building characteristics, and indoor concentration of volatile organic compounds (VOCs) in dwellings were studied. METHODS: The study comprised 88 subjects, aged 20-45 years, from the general population in Uppsala, a mid-Swedish urban community, selected by stratified random sampling. Room temperature, air humidity, respirable dust, carbon dioxide (CO2), VOCs, formaldehyde, and house dust mites were measured in the homes of the subjects. They underwent a structured interview, spirometry, peak expiratory flow (PEF) measurements at home, methacholine provocation test for bronchial hyperresponsiveness, and skin prick tests. In addition, serum concentration of eosinophilic cationic protein (S-ECP), blood eosinophil count, and total immunoglobulin E (S-IgE) were measured. RESULTS: Symptoms related to asthma were more common in dwellings with house dust mites, and visible signs of dampness or microbial growth in the building. Significant relations were also found between nocturnal breathlessness and presence of wall to wall carpets, and indoor concentration of CO2, formaldehyde, and VOCs. The formaldehyde concentration exceeded the Swedish limit value for dwellings (100 micrograms/m3) in one building, and CO2 exceeded the recommended limit value of 1000 ppm in 26% of the dwellings, showing insufficient outdoor air supply. Bronchial hyperresponsiveness was related to indoor concentration of limonene, the most prevalent terpene. Variability in PEF was related to two other terpenes; alpha-pinen and delta-karen. CONCLUSION: Our results suggest that indoor VOCs and formaldehyde may cause asthma-like symptoms. There is a need to increase the outdoor air supply in many dwelling, and wall to wall carpeting and dampness in the building should be avoided. Improved indoor environment can also be achieved by selecting building materials, building construction, and indoor activities on the principle that the emission of volatile organic compounds should be as low as reasonably achievable, to minimise symptoms related to asthma due to indoor air pollution.

Adult↗

Inhibition of human cytochromes P450 by components of Ginkgo biloba.

The extraction, isolation and characterization of 29 natural products contained in Ginkgo biloba have been described, which we have now tested for their in-vitro capacity to inhibit the five major human cytochrome P450 (CYP) isoforms in human liver microsomes. Weak or negligible inhibitory activity was found for the terpene trilactones (ginkgolides A, B, C and J, and bilobalide), and the flavonol glycosides. However 50% inhibitory activity (IC50) was found at concentrations less than 10 microg L(-1) for the flavonol aglycones (kaempferol, quercetin, apigenin, myricetin, tamarixetin) with CYP1A2 and CYP3A. Quercetin, the biflavone amentoflavone, sesamin, as well as (Z,Z)-4,4'-(1,4-pentadiene-1,5-diyl)diphenol and 3-nonadec-8-enyl-benzene-1,2-diol, were also inhibitors of CYP2C9. The IC50 of amentoflavone for CYP2C9 was 0.019 microg mL(-1) (0.035 microM). Thus, the principal components of Ginkgo biloba preparations in clinical use (terpene trilactones and flavonol glycosides) do not significantly inhibit these human CYPs in-vitro. However, flavonol aglycones, the biflavonol amentoflavone and several other non-glycosidic constituents are significant in-vitro inhibitors of CYP. The clinical importance of these potential inhibitors will depend on their amounts in ginkgo preparations sold to the public, and the extent to which their bioavailability allows them to reach the CYP enzymes in-situ.

Cytochrome P-450 Enzyme Inhibitors↗

Inhibitors of protein: geranylgeranyl transferases.

The enzyme protein:geranylgeranyl transferase-1 (PGGT-1 or GGTase-I) catalyzes the geranylgeranylation of cysteine residues near the C-termini of a variety of proteins, including most monomeric GTP binding precursor proteins belonging to the Rho, Rac and Rap subfamilies. These proteins are involved in signaling pathways controlling important processes such as cell differentiation and growth. In the framework of the development of therapeutics against disorders associated with aberrant cell proliferation, the interference with these signal transduction cascades has been a major focus of investigation. For instance inhibitors of PGGT-1 have shown promise in the treatment of cancer, smooth muscle hyperplasia as well as parasitic infections, such as malaria. In this review, structural and mechanistic aspects of the protein:geranylgeranyl transferases are discussed as well as their importance with respect to the terpene metabolism. An extensive summary of reported inhibitors of PGGT-1, classified as natural products, peptide substrate (Ca(1)a(2)L box), terpene substrate (geranylgeranyl pyrophosphate) and others, is presented. The few known inhibitors of the other geranylgeranylating enzyme, protein:geranylgeranyl transferase-2 (PGGT-2), are also included.

Alkyl and Aryl Transferases↗

Transdermal permeation modulation by cyclodextrins: a mechanistic study.

The purpose of this study was to investigate permeation modulation by beta- and 2-hydroxypropyl-beta-cyclodextrins (beta-CD and HP-beta-CD, respectively) alone and complexed with penetration enhancers for the test drugs 5-fluorouracil and estradiol through human skin, and to probe the value of the CDs in a barrier cream against toluene exposure. Methods include phase solubility studies, permeation experiments, and thermal analysis of stratum corneum; inclusion complexes were characterized by Karl Fischer titrimetry, infrared spectroscopy, and thermal analysis. Results show that complexes of terpenes or toluene with beta-CD were insoluble, whereas those with HP-beta-CD were soluble. The CDs did not enhance flux of either the polar or lipophilic drugs through skin; estradiol permeation was reduced following membrane pretreatment with either CD. Complexation of the lipophilic terpenes with the CDs reduced enhancer efficacy. When formulated into a barrier ointment both CDs, but particularly beta-CD, retarded toluene permeation through the skin and delayed the onset of maximum flux. It is concluded that the CDs themselves are not penetration enhancers for 5-fluorouracil or estradiol in human skin, and that they may be usefully incorporated into a barrier formulation to reduce percutaneous absorption of toxic materials on occupational exposure.

2-Hydroxypropyl-beta-cyclodextrin↗

Aroma compound production in cheese curd by coculturing with selected yeast and bacteria.

The microorganisms involved in cheese ripening produce various volatile compounds and induce typical flavors that contribute to cheese variety. To investigate aroma compound generation of cheese microflora, we used a dynamic headspace-gas chromatography-mass spectrometry analysis. To obtain good sensitivity and repeatability of quantification, dynamic headspace conditions and sample preparation were first optimized and led to an extraction set up in which samples were heated at 60 degrees C and diluted with water without pH adjustment. Then three different yeasts and three Geotrichum candidum commonly used in mold surface ripened cheeses were studied in pure culture in a cheese model medium. Thirty-nine cocultures of these three yeasts, the three G. candidum, and five bacteria were studied in the same medium to assess the interaction between microorganisms on aroma compound production. Twenty-four volatile compounds belonging to different chemical classes (alcohols, aldehydes, esters, sulfides, terpenes) were identified and quantified. Yeasts and especially Kluyveromyces lactis produced large amounts of alcohols, aldehydes, esters, and terpenes when cultured alone or in association. Geotrichum candidum and especially G. candidum strain G3 generated the largest amount of sulfides when cultured alone or in association. Finally, bacteria also produced aroma compounds but, except for Brevibacterium linens strain B5, which produced dimethyl trisulfide and ketones, no specific trend in the production of particular aroma compounds could be evidenced.

Bacteria↗

Formaldehyde exposure and respiratory and related cancers. A case-referent study among Finnish woodworkers.

A case-referent study was undertaken to investigate the associations between formaldehyde exposure and respiratory and related cancers. Fifty-seven such cancers from a retrospective cohort of male woodworkers formed the case group. They were matched by year of birth with 171 referents. Exposure to formaldehyde was assessed with job-exposure matrices. The median of the time-weighted average concentration was about 1 ppm, and the mean duration of exposure was 10 years among the exposed. Odds ratios (OR) were calculated for formaldehyde exposure (1.44), peak exposure to formaldehyde (1.26), and exposure to formaldehyde-containing wood dust (1.22). None of the values exceeded unity with statistical significance. Allowance was also made for a 10-year period from the onset of exposure. Birth year, cigarette smoking and exposure to wood dust, chlorophenols, pesticides, and terpenes were controlled by stratification. The adjusted ORs did not change appreciably. The highest OR was 1.95 for formaldehyde exposure without allowance for minimum latency, adjusted for exposure to terpenes. No exposure-response relation was observed for the level, duration, or dose (ppm-years) of formaldehyde exposure. The result is nonpositive and may be explained by absence of effect, by too short a follow-up, or by insufficient power for detecting a mild excess risk.

Adult↗

Ginkgo biloba--an appraisal.

Ginkgo biloba has been used in traditional Chinese medicine for about 5000 years. A standardized preparation, EGb 761 has been recently prepared. The pharmacologically active constituents, flavonol glycosides and the terpene lactones are standardized. The terpene lactones comprise of ginkgolides A, B, C and bilobalides. The extract scavenges excess free radicals and pretreatment with EGb 761 reduces damage by free radicals in patients undergoing coronary bypass surgery. The action of platelet activating factor is antagonized and platelet aggregation is reduced. Blood flow is increased. Release of prostacyclines and nitric oxide was shown to be stimulated. Ginkgo biloba has been found to be useful in the treatment of Alzheimers disease and cognitive impairment. EGB 761 has shown beneficial effect in aging and mild cognitive impairment. Bilobalide has been shown to be protective against glutamate-induced excitotoxic neuronal death. Early studies indicate a potential role in age-related macular degeneration and some types of glaucoma. Anticancer action is related to antioxidant, anti-angiogenic and gene regulatory actions. Ginkgo biloba has shown overall improvement in about 65% of patients with cerebral impairment and a similar percentage suffering from peripheral vascular diseases. A recent study suggested that phytoestrogens in Ginkgo biloba may have a role as alternative hormone replacement therapy. Recent trials have not shown a beneficial effect of Ginkgo biloba in tinnitus and acute mountain sickness. Ginkgo biloba increased the bioavailability of diltiazem. The extract has been shown to protect against doxorubicin-induced cardiotoxicity and gentamicin-induced nephrotoxicity in animals. Ginkgo biloba inhibits microsomal enzymes and has a potential for drug interactions. Further studies to establish the efficacy of Ginkgo biloba are required.

Ginkgo biloba↗

Glucuronidation of 2-arylpropionic acids pirprofen, flurbiprofen, and ibuprofen by liver microsomes.

Acylglucuronide formation from the 2-arylpropionic acids pirprofen, flurbiprofen, and ibuprofen, three nonsteroidal anti-inflammatory drugs (NSAIDs), was investigated in rat liver microsomes using an HPLC method and 14C-labeled UDP-glucuronic acid as co-substrate. Pirprofen was the best substrate of UDP-glucuronosyltransferase with a Vmax/Km of 45.4, as compared with 8.0 and 1.6 for flurbiprofen and ibuprofen, respectively. Glucuronidation of the drugs was significantly increased upon treatment of rats with phenobarbital; 3-methylcholanthrene or clofibrate failed to induce the activity. At the dose of 100 mg/kg body weight for 1, 3, and 5 days, pirprofen was unable to induce its own glucuronidation. However, this treatment caused a transient increase, after 1 day, of several isoform activities monitored with 4-nitrophenol, 1-naphthol, 4-methylumbelliferone, terpenes, and testosterone as substrates. After 3 and 5 days these activities were decreased, especially when glucuronidation of 4-nitrophenol and 4-methylumbelliferone was considered, glucuronidation of the terpenes cis-myrtanol, borneol, nopol, and of testosterone being similar to control values. By contrast to clofibrate, administration of pirprofen to rats decreased bilirubin UDP-glucuronosyltransferase in a time-dependent fashion with a maximal decrease of 59% after 5 days. Treatment of rats with pirprofen also decreased markedly the formation of flurbiprofen glucuronide. Comparison of NSAID glucuronidation between several species indicated that it was most potent in monkeys, dogs, and humans. Cats were also efficient in that respect. Gunn rats, which are genetically deficient in bilirubin glucuronidation, were able to form acylglucuronides from the drugs, thus indicating that these 2-arylpropionic acids were not substrates of the bilirubin isozyme.

Aged↗

Genomic mechanism of aroma terpenoids biosynthesis in plants.

BACKGROUND: Aroma terpenoids are crucial plant secondary metabolites with physiological and commercial importance. Interestingly, both closely and distantly related species can synthesize identical aroma terpenoids. With the development of genome sequencing technology, it has become possible to elucidate the genomic mechanism underlying this phenomenon. AIM: This review highlights whole-genome data as a robust strategy for investigating the genomic mechanism of aroma terpenoids biosynthesis in plants, and provides new perspectives on the origin, evolution, and engineering of terpene synthases (TPSs). This aims to significantly benefit plant breeding and enhance suitability for industrial production. KEY SCIENTIFIC CONCEPTS OF REVIEW: Genomic mechanism of aroma terpenoids biosynthesis in plant genomes is the genetic and evolutionary dynamics. We elaborate the genomic mechanism governing the biosynthesis of plant-derived aroma terpenoids in three dimensions: (1) Genome-wide identification and phylogenetic analyses of TPSs. The same aroma terpenoids were produced by numerous plant species with chromosome-level genomes. Based on 34 plant genomes, we identified 1643 TPSs and classified them into seven subfamilies. (2) Functional and structural basis of TPSs. We found that TPSs with identical functions in distant species exhibit low sequence similarity but conserved active cavity architectures. Conversely, functionally distinct TPSs in closely related species cluster phylogenetically but differ in active cavity structures. (3) Patterns of TPS gene origination. Comparative genomic analyses within and between species revealed three patterns enabling TPSs to acquire the same functions: tandem duplications, dispersed duplications, and genes without duplication.

Terpenes↗

CATNIP: ITS RAISON D' ETRE.

Catnip (nepetalactone) is closely related chemically to certain cyclopentanoid monoterpenes recently isolated from insects, and it shares with some of these terpenes an ability to repel insects. It is suggested that the adaptive function of catnip is to protect the plants that produce it against phytophagous insects.

Animals↗

Liquid chromatography/electrospray mass spectrometry of bioactive terpenoids in Ginkgo biloba L.

Standardized extracts of Ginkgo biloba leaves are mainly used in the treatment of peripheral and celebral circulation disorders, and also as a remedy against asthma, coughs, bladder inflammation, blenorrhagia and alcohol abuse. The leaf extracts contain biflavones, flavonol glycosides and terpene lactones. This paper reports a method based on liquid chromatography coupled with electrospray mass spectrometry for the analysis of terpenoids in G. biloba extracts. This method allows the rapid isocratic separation of underivatized ginkgolides (GA, GB, GC and GJ) and bilobalide at very low levels (10 pg on the column) and their quantitative detection by external standardization with relative standard deviations of 3 and 5% for intra- and inter-day analyses, respectively.

Chromatography, Liquid↗

A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential.

The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat kidney and urinary bladder disturbances, intestinal infections, diabetes, and hepatitis B. In recent years, the interest in the plants has increased considerably. Substantial progress on their chemistal and pharmacological properties, as well as a few clinical studies of some Phyllanthus species have been made. This review discusses the current knowledge of their chemistry, the in vitro and in vivo pharmacological, biochemical, and clinical studies carried out on the extracts, and the main active constituents isolated from different species of plants of the genus Phyllanthus. These studies carried out with the extracts and purified compounds from these plants support most of their reported uses in folk medicine as an antiviral, in the treatment of genitourinary disorders, and as antinociceptive agents. However, well-controlled, double-binding clinical trials are lacking. Several compounds including alkaloids, flavonoids, lignans, phenols, and terpenes were isolated from these plants and some of them interact with most key enzymes. Together this data strongly supports the view that the plants belonging to the genus Phyllanthus have potential beneficial therapeutic actions in the management of hepatitis B, nefrolitiase, and in painful disorders.

Alkaloids↗

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

Alkyl and Aryl Transferases↗

High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli.

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.

Antimalarials↗

Gallstone clearance: a randomized study of extracorporeal shock wave lithotripsy and chemical dissolution.

Following extracorporeal shock wave lithotripsy it is not known whether gallstone fragments are cleared from the gallbladder without the use of oral dissolution therapy. To assess the efficacy of lithotripsy and dissolution therapy, alone or in combination, 35 patients were randomized to one of three treatment groups: lithotripsy alone, dissolution therapy alone or combined lithotripsy and dissolution therapy. All patients had symptomatic gallstones, functioning gallbladders and comparable stone profiles. Lithotripsy was administered using a piezoelectric lithotripter. Dissolution therapy consisted of combined bile acid and terpene. Clearance was assessed at 6 months using ultrasound and oral cholecystography. Patients with less than 50 per cent stone clearance at the end of 6 months were considered failures. The number of patients with total or partial clearance in the combined group (7/10) was significantly greater than those in the lithotripsy alone group (0/10, P less than 0.002). Gallstone clearance following lithotripsy appears to be dependent upon dissolution therapy.

Adult↗

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus.

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.

Acrylates↗

Evaluation of the mutagenicity of beta-myrcene in mammalian cells in vitro.

The genotoxicity of the terpene beta-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 micrograms/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt-locus in V79-cells. Tests with and without S9-mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE-inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.

Acyclic Monoterpenes↗