Glucocorticoid hormones in relation to environmental exposure to bisphenols and multiclass pesticides among middle aged-women: Results from hair analysis.

Bisphenols and pesticides have been shown to alter circulating glucocorticoids levels in animals, but there is limited human data. Moreover, measurements from biological fluids may not be able to reflect long-term status of non-persistent pollutants and glucocorticoids due to the high variability in their levels. Using hair analysis, we examined the associations between glucocorticoid hormones and environmental exposure to multi-class organic pollutants among a healthy female population aged 25-45 years old. Concentrations of four glucocorticoids, four polychlorinated biphenyl congeners (PCBs), seven polybrominated diphenyl ether congeners (PBDEs), two bisphenols and 140 pesticides and their metabolites were measured in hair samples collected from 196 Chinese women living in urban areas. Due to the low detection frequency of some pollutants, associations were explored only on 54 pollutants, i.e. PCB 180, bisphenol A, bisphenol S and 51 pesticides and their metabolites. Using stability-based Lasso regression, there were associations of cortisol, tetrahydrocortisol, cortisone, and tetrahydrocortisone with 14, 10, 13 and 17 biomarkers of exposure to pollutants, respectively, with bisphenol S, p,p'-dichlorodiphenyldichloroethylene, diethyl phosphate, 3,5,6-trichloro-2-pyridinol, thiamethoxam, imidacloprid, fipronil, tebuconazole, trifluralin, pyraclostrobin and 1-(3,4-dichlorophenyl)-3-methylurea being associated with at least three of the four hormones. There were also associations between cortisone/cortisol molar ratio and pollutants, namely dimethyl phosphate, 3-methyl-4-nitrophenol, carbofuran, λ-cyhalothrin, permethrin, fipronil, flusilazole, prometryn and fenuron. Some of these relationships were confirmed by single-pollutant linear regression analyses. Overall, our results suggest that background level of exposure to bisphenols and currently used pesticides may interfere with the glucocorticoid homeostasis in healthy women.

Association between Environmental Exposure to Multiclass Organic Pollutants and Sex Steroid Hormone Levels in Women of Reproductive Age.

Organic pollutant exposure may alter sex steroid hormone levels in both animals and humans, but studies on mixture effects have been lacking and mainly limited to persistent organic pollutants, with few hormones being investigated. Moreover, measurements from a single blood or urine sample may not be able to reflect long-term status. Using hair analysis, here, we evaluated the relationship between multiclass organic pollutants and sex steroid hormones in 196 healthy Chinese women aged 25-45 years. Associations with nine sex steroid hormones, including progesterone, androstenedione (AD), testosterone (T), estrone (E1), and 17ß-estradiol (E2), and eight related hormone ratios were explored on 54 pollutants from polychlorinated biphenyl (PCB), pesticide, and bisphenol families using stability-based Lasso regression analysis. Our results showed that each hormone was associated with a mixture of at least 10 examined pollutants. In particular, hair E2 concentration was associated with 19 pollutants, including γ-hexachlorocyclohexane, propoxur, permethrin, fipronil, mecoprop, prochloraz, and carbendazim. There were also associations between pollutants and hormone ratios, with pentachlorophenol, dimethylthiophosphate, 3-phenoxybenzoic acid, and flusilazole being related to both E1/AD and E2/T ratios. Our results suggest that exposure to background levels of pesticides PCB180 and bisphenol S may affect sex steroid hormone homeostasis among women of reproductive age.

Investigating the biosynthesis of Sch-642305 in the fungus Phomopsis sp. CMU-LMA.

Sch-642305 is an unusual bicyclic 10-membered macrolide produced by the filamentous fungus Phomopsis sp. CMU-LMA for which no biosynthetic evidence exists. Here, we generate a draft genome sequence of the producing organism and discover the biosynthetic gene cluster responsible for formation of Sch-642305. Targeted gene disruptions together with reconstitution of the pathway in the heterologous host Aspergillus oryzae dissect key chemical steps and shed light on a series of oxidoreductions occuring in the pathway.

H3K4 trimethylation by CclA regulates pathogenicity and the production of three families of terpenoid secondary metabolites in Colletotrichum higginsianum.

The role of histone 3 lysine 4 (H3K4) methylation is poorly understood in plant pathogenic fungi. Here, we analysed the function of CclA, a subunit of the COMPASS complex mediating H3K4 methylation, in the brassica anthracnose pathogen Colletotrichum higginsianum. We show that CclA is required for full genome-wide H3K4 trimethylation. The deletion of cclA strongly reduced mycelial growth, asexual sporulation and spore germination but did not impair the morphogenesis of specialized infection structures (appressoria and biotrophic hyphae). Virulence of the ΔcclA mutant on plants was strongly attenuated, associated with a marked reduction in appressorial penetration ability on both plants and inert cellophane membranes. The secondary metabolite profile of the ΔcclA mutant was greatly enriched compared to that of the wild type, with three different families of terpenoid compounds being overproduced by the mutant, namely the colletochlorins, higginsianins and sclerosporide. These included five novel molecules that were produced exclusively by the ΔcclA mutant: colletorin D, colletorin D acid, higginsianin C, 13-epi-higginsianin C and sclerosporide. Taken together, our findings indicate that H3K4 trimethylation plays a critical role in regulating fungal growth, development, pathogenicity and secondary metabolism in C. higginsianum.

Deleting a Chromatin Remodeling Gene Increases the Diversity of Secondary Metabolites Produced by Colletotrichum higginsianum.

Colletotrichum higginsianum is the causal agent of crucifer anthracnose disease, responsible for important economic losses in Brassica crops. A mutant lacking the CclA subunit of the COMPASS complex was expected to undergo chromatin decondensation and the activation of cryptic secondary metabolite biosynthetic gene clusters. Liquid-state fermentation of the Δ cclA mutant coupled with in situ solid-phase extraction led to the production of three families of compounds, namely, colletorin and colletochlorin derivatives with two new representatives, colletorin D (1) and colletorin D acid (2), the diterpenoid α-pyrone higginsianin family with two new analogues, higginsianin C (3) and 13- epi-higginsianin C (4), and sclerosporide (5) coupling a sclerosporin moiety with dimethoxy inositol.

Isolation of the antibiotic methyl (R,E)-3-(1-hydroxy-4-oxocyclopent-2-en-1-yl)-acrylate EA-2801 from Trichoderma atroviridae.

The endophytic Trichoderma atroviridae UB-LMA was isolated as a symbiont of Taxus baccata and analyzed for its antimicrobial activity. By applying an original approach consisting of solid-state cultivation coupled with solid-phase extraction, a new methyl (R,E)-3-(1-hydroxy-4-oxocyclopent-2-en-1-yl)-acrylate derivative named EA-2801 (1) was isolated together with the previously reported isonitrin A and dermadin methyl ester. The chemical structure of 1 was determined by NMR and MS. Compound 1 showed antimicrobial activity against a panel of Gram-positive and -negative bacteria.

Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines.

The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studiedagainst three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chormatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-ß-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer.

Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata.

Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-state fermentation coupled to in situ solid phase extraction (SPE) was applied, and four compounds were discovered. Compounds 2-4 belong to the harziane tetracyclic diterpene family. Bicylic compound 1 may represent the biosynthetic precursor of this scarce family of compounds.

Bioactive polyketides isolated from agar-supported fermentation of Phomopsis sp. CMU-LMA, taking advantage of the scale-up device, Platotex.

Phomopsis sp. CMU-LMA was cultivated on agar-supported fermentation (Ag-SF) using the scale-up prototype Platotex. In total nine compounds were isolated from the ethyl acetate extract of the culture. Among them, compounds LMA-P1, Sch-642305, DHTO and LMA-P2 had already been reported in our previous work on liquid state fermentation. The trihydroxybenzene lactone cytosporone D and dothiorelone A has been recently isolated from Phomopsis and Magnaporthe species. In addition, three compounds were isolated consisting in the reduced methoxy derivative of Sch-642305 (1), a hydroxylated derivative of LMA-P2 (2) and a linear ethyl ester polyketide (3) similar to the previously reported LMA-P3. Antimicrobial activity and inhibition of Escherichia coli DnaG primase were investigated. Cytosporone D inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC50 of 0.25 mM.

Isolation and characterization of unusual hydrazides from Streptomyces sp. impact of the cultivation support and extraction procedure.

Three novel hydrazides, geralcins C-E (1-3), were isolated from Streptomyces sp. LMA-545, together with MH-031 and geralcins A and B. This unusual family of compounds was isolated from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. The use of such neutral resin during the cultivation step allowed the specific adsorption of microbial secondary metabolites, avoiding any contamination of the crude extracts by the constituents of the culture medium. The trapped compounds were eluted from the resin with methanol, and their structures elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Molecular modeling calculations were applied in order to support structural attributions. No antimicrobial, cytotoxic, or DnaG-inhibition activities were detected for geralcins D and E. Geralcin C has no antimicrobial activity but exhibited an IC(50) of 0.8 µM against KB and HCT116 cancer cell lines. Furthermore, geralcin C inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC(50) of 0.7 mM.

Application of solid-phase extraction to agar-supported fermentation.

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m(2) scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a "solid-solid extraction" (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.

Biotransformation of natural compounds: unexpected thio conjugation of Sch-642305 with 3-mercaptolactate catalyzed by Aspergillus niger ATCC 16404 cells.

Sch-642305 is produced by the endophytic fungi Phomopsis sp. CMU-LMA and exhibits both antimicrobial and cytotoxic activities. The incubation of Sch-642305 with Aspergillus niger ATCC 16404 resting cells leads to two unexpected thio conjugates. Compound (1) is formed by the addition of the cysteine metabolite 3-mercaptolactate to the double bond of Sch-642305. Compound (1) undergoes an intramolecular rearrangement to give compound (2), which contains two rings: a five-membered hydroxylactone ring and a five-membered thiophene ring. The absolute configuration of compound (1) is similar to that of the parent compound, but the configuration of the mercaptolactate side-chain was not determined. The absolute configuration of compound (2) was deduced from the crystal structure and confirmed by the anomal effect of the sulfur atom. To the best of our knowledge, this is the first time such a conjugation rearrangement reactions were observed. The biological significance and the reaction mechanisms are discussed. Compound (1) exhibits a weak antimicrobial activity against Gram-positive bacteria, whereas derivatives (1) and (2) showed an IC50 of 1 and 1.2 µM, respectively, against colonic epithelial cancer cells.

Isolation, structure elucidation and biological activity of metabolites from Sch-642305-producing endophytic fungus Phomopsis sp. CMU-LMA.

Eight polyketide compounds were isolated from the cultivation broth of Phomopsis sp. CMU-LMA. We have recently described LMA-P1, a bicyclic 10-membered macrolide, obtained as a bioconversion derivative of Sch-642305, the major compound isolated in this study. Benquinol is the ethyl ester derivative of the 13-dihydroxytetradeca-2,4,8-trienoic acid produced by Valsa ambiens. This compound is concomitantly produced with the 6,13-dihydroxytetradeca-2,4,8-trienoic acid (DHTTA) previously isolated from Mycosphaerellarubella. The absolute configuration of the new compound, (2R,3R,4S,5R)-3-hydroxy-2,4-dimethyl-5-[(S,Z)-3-methylpentenyl]-tetrahydro-pyranone LMA-P2 was confirmed by X-ray crystallography. The δ-lactone 2,3-dihydroxytetradecan-5-olide (DHTO) was previously isolated from Seiridium unicorne. This compound may form through the cyclization of the methyl-2,3,5-trihydroxytridecanoate LMA-P3, a new linear polyketide isolated in this study. Benquoine, a new 14-membered lactone generated from the cyclization of benquinol, is proposed as the key precursor for the biosynthesis of Sch-642305. Antimicrobial activity and cancer cell viability inhibition by the new compounds were investigated. Benquoine exhibits antimicrobial activity against Gram positive bacteria, and cytotoxicity against HCT-116 cancer cell line.

Biotransformation of natural compounds. Oxido-reduction of Sch-642305 by Aspergillus ochraceus ATCC 1009.

Sch-642305 is the major compound produced by the endophytic fungi Phomopsis sp. CMU-LMA. Incubation of Sch-642305 with Aspergillus ochraceus ATCC 1009 resting cells leads to three new derivatives through an oxido-reduction of the six-membered ring of the molecule. Reduction of the double bound leads to compound (1), which subsequently undergoes carbonyl reduction to (2) and ring hydroxylation to (3). According to the previously solved crystal structure of Sch-642305 coupled with (1)H NMR NOE correlation and the crystal structure of compound 1, the absolute configurations of the new derivatives were established. In contrast to the parent compound Sch-642305, compound (1) exhibits antimicrobial activity against gram-negative bacteria. Furthermore, while all derivatives exhibit cytotoxic activity against various cancer cell lines, compound (2) achieved an IC(50) of 4 nM against human myelogenous leukemia K 562, compared to 20 nM for the parent Sch-642305.

Platotex: an innovative and fully automated device for cell growth scale-up of agar-supported solid-state fermentation.

Among various factors that influence the production of microbial secondary metabolites (MSM), the method of cultivation is an important one that has not been thoroughly investigated. In order to increase microbial throughput and simplify the extraction and workup steps, we performed a study to compare liquid-state fermentation (LSF) with agar-supported solid-state fermentation (AgSF). We found that AgSF is not only more suitable for our applications but offers, for some microbial strains, a higher yield and broader diversity of secondary metabolites. The main limitation of AgSF is the lack of a system to allow production scale-up. In order to overcome this obstacle we developed Platotex, an original fermentation unit offering 2 m(2) of cultivation surface that combines automatic sterilization, cultivation, and drying steps. Platotex is also able to support both LSF and solid-state fermentation (SSF). Platotex conforms to international security and quality requirements and benefits from total remote automation through industrial communication and control standards.