Herbivory by an Outbreaking Moth Increases Emissions of Biogenic Volatiles and Leads to Enhanced Secondary Organic Aerosol Formation Capacity.

In addition to climate warming, greater herbivore pressure is anticipated to enhance the emissions of climate-relevant biogenic volatile organic compounds (VOCs) from boreal and subarctic forests and promote the formation of secondary aerosols (SOA) in the atmosphere. We evaluated the effects of Epirrita autumnata, an outbreaking geometrid moth, feeding and larval density on herbivore-induced VOC emissions from mountain birch in laboratory experiments and assessed the impact of these emissions on SOA formation via ozonolysis in chamber experiments. The results show that herbivore-induced VOC emissions were strongly dependent on larval density. Compared to controls without larval feeding, clear new particle formation by nucleation in the reaction chamber was observed, and the SOA mass loadings in the insect-infested samples were significantly higher (up to 150-fold). To our knowledge, this study provides the first controlled documentation of SOA formation from direct VOC emission of deciduous trees damaged by known defoliating herbivores and suggests that chewing damage on mountain birch foliage could significantly increase reactive VOC emissions that can importantly contribute to SOA formation in subarctic forests. Additional feeding experiments on related silver birch confirmed the SOA results. Thus, herbivory-driven volatiles are likely to play a major role in future biosphere-vegetation feedbacks such as sun-screening under daily 24 h sunshine in the subarctic.

Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 µM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

Exposure during pregnancy to glycol ethers and chlorinated solvents and the risk of congenital malformations.

BACKGROUND: Exposure to solvents during pregnancy has long been suspected of increasing the risk of congenital malformations, but the lack of prospective assessment of specific solvent exposures has prevented definitive conclusions. METHODS: In a cohort of 3421 pregnant women in Brittany (2002-2006), occupational solvent exposure was assessed from self-report during pregnancy and from a job-exposure matrix. Congenital malformations were diagnosed among live births, stillbirths, and medical pregnancy terminations. In a nested case-control sample, urinary concentrations of 10 metabolites of glycol ethers and chlorinated solvents were measured in maternal samples collected during early pregnancy (n = 79 cases, 580 controls). RESULTS: Dose-response trends linked occupational solvent exposure (both self-reported and based on a job-exposure matrix) to the risk of major congenital malformations--especially oral clefts, urinary tract malformations, and male genital malformations. Detection of some glycol ether metabolites and trichloroacetic acid in urine was associated with increased risks of oral clefts and of urinary tract and limb defects. CONCLUSIONS: This prospective study, using three independent methods of exposure assessment, suggests several specific associations between solvent exposure during early pregnancy and congenital malformations. Results based on urinary biomarkers, although limited by small numbers, identify work situations that require further investigation.

Molecular dynamics simulations for human CAR inverse agonists.

Constitutive androstane receptor (CAR), along with pregnane x receptor (PXR), is an important metabolic sensor in the hepatocytes. Like all other nuclear receptors (NRs), CAR works in concert with coregulator proteins, coactivators, and corepressors which bind to the NRs. The main basis for the receptor to distinguish between coactivators and corepressors is the position of the C-terminal helix 12 (H12), which is determined by the bound NR ligand. CAR, having constitutive activity, can be repressed or further activated by its ligands. Crystal structure of human CAR bound to an agonist and a coactivator peptide is available, but no structural information on an inverse agonist-bound human CAR and a corepressor exists. In our previous molecular dynamics (MD) studies, no corepressor peptide was included. Therefore, probably due to the strong interactions which keep the relatively short H12 of CAR in the active position, the structural changes elicited by inverse agonists were very subtle, and H12 of CAR seemed to more or less retain its active conformation. Here, we have run a series of MD simulations to study the movement of H12 in the presence of both activating and repressing ligands as well as a corepressor peptide. The presence of the corepressor on the coregulator surface of CAR induced a clear shift of H12 of the inverse agonists-bound CAR. In general, H12 moved toward H10 and not away from the ligand binding domain, as seen in some other NRs. However, H12 of CAR is short enough that this movement seems to be adequate to accommodate the binding of the corepressor.

Urinary biomarkers of exposure to glycol ethers and chlorinated solvents during pregnancy: determinants of exposure and comparison with indirect methods of exposure assessment.

OBJECTIVES: To describe urine levels of metabolites of glycol ethers and chlorinated solvents in a sample of pregnant women from the general population, to study their occupational and non-occupational determinants and to compare them with the results of indirect assessment methods of solvent exposure. METHODS: A sample of 451 pregnant women was randomly selected from a general population cohort. At inclusion, the women in this sample completed a self-administered questionnaire about their social and medical characteristics, occupation and exposure to different products at work and in non-occupational activities. Occupational exposure to solvents was assessed from the woman's self-report and from a job-exposure matrix. Eight alkoxycarboxylic acids and trichloroacetic acid and trichloroethanol were measured with chromatography in urine samples collected at inclusion. Associations between metabolite levels and job titles, exposure to products used at work, and solvent exposure were studied. RESULTS: The different glycol ether metabolites were detected in 5.3%-96.4% of the urine samples, trichloroacetic acid in 6.4% and trichloroethanol in 5.5%. Nurses had butoxyacetic acid and phenoxyacetic acid in their urine most often, whereas methoxyethoxyacetic acid was the most frequent among nursing aides. Among cleaners, ethoxyacetic acid and ethoxyethoxyacetic acid were the most frequent. The occupation of hairdresser was associated with urinary excretion of ethoxyacetic acid, ethoxyethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid. Among the women classified as exposed to solvents, the agents identified most often were ethoxyacetic acid, ethoxy-ethoxyacetic acid, butoxyacetic acid, phenoxyacetic acid, trichloroacetic acid and trichloroethanol. Ethoxyethoxyacetic acid was the only metabolite associated with non-occupational exposure. CONCLUSIONS: Metabolites of glycol ethers and chlorinated solvents were present at low levels in the urine of pregnant women. Most metabolites were associated with occupational exposure.

Birch (Betula spp.) leaves adsorb and re-release volatiles specific to neighbouring plants--a mechanism for associational herbivore resistance?

Plant-emitted semi-volatile compounds have low vaporization rates at 20-25 degrees C and may therefore persist on surfaces such as plant foliage. The passive adsorption of arthropod-repellent semi-volatiles to neighbouring foliage could convey associational resistance, whereby a plant's neighbours reduce damage caused by herbivores. We found that birch (Betula spp.) leaves adsorb and re-release the specific arthropod-repelling C(15) semi-volatiles ledene, ledol and palustrol produced by Rhododendron tomentosum when grown in mixed association in a field setup. In a natural habitat, a higher concentration of ledene was released from birches neighbouring R. tomentosum than from birches situated > 5 m from R. tomentosum. Emission of alpha-humulene, a sesquiterpene synthesized by both Betula pendula and R. tomentosum, was also increased in R. tomentosum-neighbouring B. pendula. In assessments for associational resistance, we found that the polyphagous green leaf weevils (Polydrusus flavipes) and autumnal moth (Epirrita autumnata) larvae both preferred B. pendula to R. tomentosum. P. flavipes also preferred birch leaves not exposed to R. tomentosum to leaves from mixed associations. In the field, a reduction in Euceraphis betulae aphid density occurred in mixed associations. Our results suggest that plant/tree species may be protected by semi-volatile compounds emitted by a more herbivore-resistant heterospecific neighbour.

Synthesis and biological evaluation of phenolic 4,5-dihydroisoxazoles and 3-hydroxy ketones as estrogen receptor alpha and beta agonists.

In this work, 52 diphenyl-4,5-dihydroisoxazoles and -3-hydroxy ketones were prepared and their estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) activities were explored in order to systematize and maximize their biological activity. The biological activity was firstly screened by using ERE reporter assay to find out how aromatic hydroxylation and methylation of the chiral centers of the compounds affect the ability of ER to mediate biological responses. For selected 19 compounds, the relative binding affinities (RBA, relative to 3,17beta-estradiol) and ability to induce transcription of primary E2 target gene pS2 in human MCF-7 breast cancer cells were determined. In the reporter assay, many compounds showed even stronger activity than E2 and some of them showed RBA larger than 1%. The highest RBAs were determined for the enantiomers of 1-hydroxy-6-(4-hydroxy-phenyl)-1-phenyl-hexan-3-one (50a and 50b). Isomer 50a showed high binding affinity both to ERalpha (with RBA approximately 200%) and ERbeta (with RBA approximately 60%), while the RBAs of 50b were ca. 40% of those. Some of the other compounds (with RBA approximately 1-16%) showed also notable ERalpha binding selectivity. When four most promising ligands (50a, 50b, 45a, and 45b) were studied with respect to their ability to induce the transcription of primary E2 target gene pS2, the compounds acted as agonists or partial agonists. Computer modeling was used to predict receptor binding conformations and to rationalize the RBA differences of the compounds.

Synthesis and evaluation of estrogen agonism of diaryl 4,5-dihydroisoxazoles, 3-hydroxyketones, 3-methoxyketones, and 1,3-diketones: a compound set forming a 4D molecular library.

In this paper, the preparation and systematic evaluation of estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) activities of some diaryl-1,3-diones and their synthetic intermediates, diaryl-4,5-dihydroisoxazoles, diaryl-3-hydroxyketones, diaryl-3-methoxyketones, and diaryl-2-(dimethyl-lambda 4-sulfanylidene)-1,3-diones, is described. The set of 72 compounds constitutes a general schematic structure aryl1-linker1-spacer-linker2-aryl2, where the linker1-spacer-linker2 length varies between 4 and 8 carbons. The set of compounds was applied here to map and explore the active sites of subtypes ER alpha and ER beta. The highest activities were obtained with dihydroisoxazole and hydroxyketone spacers, but even the most flexible diones with unsubstituted aryl groups showed some agonism. Most compounds were found to be ER alpha selective or to activate both receptors, but in some cases we saw also clearly stronger ER beta activation.

Improved assays for xenosensor activation based on reverse transfection.

Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies.

Synthesis and biological evaluation of second-generation tropanol-based androgen receptor modulators.

To circumvent antiandrogen resistance in prostate cancer, antiandrogens effective for both the androgen receptor (AR) and AR mutants are required. The AR antagonists in this study originate from previous findings, which showed that subtle differences in substitution pattern lead to a conformational change that alters the ligand activity, rendering an agonist to an antagonist. We have identified small yet potent tropanol-based ligands possessing significant antiandrogenic activity with both wild-type AR and the two most common AR ligand binding domain (LBD) mutants.

Preclinical pharmacology of FL442, a novel nonsteroidal androgen receptor modulator.

The preclinical profiles of two most potent compounds of our recently published cycloalkane[d]isoxazole pharmacophore-based androgen receptor (AR) modulators, FL442 (4-(3a,4,5,6,7,7a-hexahydro-benzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile) and its nitro analog FL425 (3-(4-nitro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole), were explored to evaluate their druggability for the treatment of AR dependent prostate cancer. The studies revealed that both compounds are selective to AR over other closely related steroid hormone receptors and that FL442 exhibits equal inhibition efficiency towards the androgen-responsive LNCaP prostate cancer cell line as the most widely used antiandrogen bicalutamide and the more recently discovered enzalutamide. Notably, FL442 maintains antiandrogenic activity with enzalutamide-activated AR mutant F876L. In contrast to bicalutamide, FL442 does not stimulate the VCaP prostate cancer cells which express elevated levels of the AR. Distribution analyses showed that [(14)CN]FL442 accumulates strongly in the mouse prostate. In spite of its low plasma concentration obtained by intraperitoneal administration, FL442 significantly inhibited LNCaP xenograft tumor growth. These findings provide a preclinical proof for FL442 as a promising AR targeted candidate for a further optimization.

Urinary glycol ether metabolites in women and time to pregnancy: the PELAGIE cohort.

BACKGROUND: Glycol ethers are present in a wide range of occupational and domestic products. Animal studies have suggested that some of them may affect ovarian function. OBJECTIVE: We examined the relation between women's exposure to glycol ethers and time to pregnancy. METHODS: We used chromatography coupled to mass spectrometry to measure eight glycol ether metabolites in urine samples from randomly selected women in the PELAGIE mother-child cohort who had samples collected before 19 weeks of gestation. Using time to pregnancy information collected at the beginning of the pregnancy (women were asked how many months it took for them to conceive), we estimated associations between metabolite levels and time to pregnancy in 519 women with complete data using discrete-time Cox proportional hazards models to adjust for potential confounders. RESULTS: We detected glycol ether metabolites in 6% (for ethoxyacetic acid) to 93% (for phenoxyacetic and butoxyacetic acids) of urine samples. Phenoxyacetic acid was the only metabolite with a statistically significant association with longer time to pregnancy [fecundability OR = 0.82; 95% CI: 0.63, 1.06 for the second and third quartile combined; fecundability OR = 0.70; 95% CI: 0.52, 0.95 for a fourth-quartile (≥ 1.38 mg/L) vs. first-quartile concentration (< 0.14 mg/L)]. This association remained stable after multiple sensitivity analyses. CONCLUSION: Phenoxyacetic acid, which was present in most of the urine samples tested in our study, was associated with increased time to pregnancy. This metabolite and its main parent compound, 2-phenoxyethanol, are plausible causes of decreased fecundability, but they may also be surrogates for potential coexposures to compounds frequently present in cosmetics.

Discovery of 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles as potent firefly luciferase inhibitors.

Luciferase reporter assays are commonly used in high-throughput screening methods. Here, we report new firefly luciferase (FLuc) inhibitors based on 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles, which showed up as "false positives" in a luciferase reporter gene-based assay for nuclear receptor antagonists. The inhibition was shown to be noncompetitive for both natural enzyme substrates (d-luciferin and ATP) and selective to FLuc and proven to arise from a direct interaction between the enzyme and the inhibitor. Of the 63 evaluated compounds, 28 showed significantly better inhibition potency than the well-known inhibitor resveratrol (IC(50) = 59 nM), with five compounds having distinctly subnanomolar IC(50) values. The most efficient compounds inhibited the luminescence at concentrations lower than (1)/(100) in comparison to resveratrol (lowest IC(50) = 0.26 nM) and can thus be considered to belong to the most potent FLuc inhibitors reported thus far. Overall, the novel inhibitors form a unique molecular library for structure-activity relationship (SAR) analyses.

Design, synthesis, and biological evaluation of nonsteroidal cycloalkane[d]isoxazole-containing androgen receptor modulators.

We report here the design, preparation, and systematic evaluation of a novel cycloalkane[d]isoxazole pharmacophoric fragment-containing androgen receptor (AR) modulators. Cycloalkane[d]isoxazoles form new core structures that interact with the hydrophobic region of the AR ligand-binding domain. To systematize and rationalize the structure-activity relationship of the new fragment, we used molecular modeling to design a molecular library containing over 40 cycloalkane[d]isoxazole derivatives. The most potent compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and hydroxyflutamide, respectively, in prostate cancer patients.

Ligand specificity of constitutive androstane receptor as probed by induced-fit docking and mutagenesis.

Constitutive androstane receptor (CAR, NR1I3) belongs to the nuclear receptor family of transcription factors and acts as a chemical sensor of drugs and endogenous compounds. The ligand-binding preferences of CAR are diverse, and more importantly, there are significant species differences in ligand specificity. Here, we show that while certain residues are critical for the basal activity of mouse CAR (mCAR) and/or affect the binding of all tested ligands, mutation of some ligand-binding pocket (LBP) residues (e.g., F171 and Y336) paradoxically decreased the activity of a specific ligand while increasing that of others. Comparisons to previously reported human CAR (hCAR) residues indicated that the function of key CAR residues (e.g., N175, L253) is dramatically different between species. The docking results provide some mechanistic rationale for the ability of 17alpha-ethinyl-3,17beta-estradiol (EE2) to both activate mCAR and repress hCAR.

Propylene glycol monomethyl ether occupational exposure (PGME). 4. Analysis of 2-methoxypropionic acid in urine.

OBJECTIVE: The two isomers propylene glycol monomethyl ether [PGME-alpha (1-methoxy-2-propanol, M2P) and PGME-beta (2-methoxy-1-propanol)] have different toxicities due to the different ways they are metabolised. The higher toxicity of PGME-beta has been attributed to the formation of 2-methoxypropionic acid (2-MPA) as a metabolite of primary alcohol. Six healthy male volunteers were exposed to PGME-alpha vapour (15, 50 and 95 ppm) with and without respiratory protection for 6 h, including a 30-min break. They were also exposed to PGME-alpha liquid (10% or 30% in water), via one hand, for 30 min or 1 h. Commercial products of M2P always contain a small quantity of the beta isomer, and GC analysis has shown that the product used for this human volunteer exposure contained approximately 0.3% of the beta isomer. The objective of this study was to determine the levels of 2-MPA in urine after these exposures to 99.7% PGME-alpha. METHOD: An analytical method developed by Laitinen [6] was used for the determination of 2-MPA in the urine of exposed volunteers. RESULTS: End exposure levels of 2-MPA were found to reach from 1.19 to 3.29 mg/l for inhalation and dermal exposure to PGME-alpha vapour and from under the detection limit to 2.10 mg/l for exposure of one hand in PGME-alpha liquid. 2-MPA concentrations in urine samples from a non-exposed person or from a person exposed to PGME-alpha vapour at 15 ppm (inhalation and dermal exposure) and also from a person exposed to PGME-alpha vapour up to 95 ppm with respiratory protection (dermal-only exposure) all varied from under the detection limit to 0.30 mg/l and are then not significant.