Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Scan-Free GEXRF in the Soft X-ray Range for the Investigation of Structured Nanosamples.

Scan-free grazing-emission X-ray fluorescence spectroscopy (GEXRF) is an established technique for the investigation of the elemental depth-profiles of various samples. Recently it has been applied to investigating structured nanosamples in the tender X-ray range. However, lighter elements such as oxygen, nitrogen or carbon cannot be efficiently investigated in this energy range, because of the ineffective excitation. Moreover, common CCD detectors are not able to discriminate between fluorescence lines below 1 keV. Oxygen and nitrogen are important components of insulation and passivation layers, for example, in silicon oxide or silicon nitride. In this work, scan-free GEXRF is applied in proof-of-concept measurements for the investigation of lateral ordered 2D nanostructures in the soft X-ray range. The sample investigated is a Si3N4 lamellar grating, which represents 2D periodic nanostructures as used in the semiconductor industry. The emerging two-dimensional fluorescence patterns are recorded with a CMOS detector. To this end, energy-dispersive spectra are obtained via single-photon event evaluation. In this way, spatial and therefore angular information is obtained, while discrimination between different photon energies is enabled. The results are compared to calculations of the sample model performed by a Maxwell solver based on the finite-elements method. A first measurement is carried out at the UE56-2 PGM-2 beamline at the BESSY II synchrotron radiation facility to demonstrate the feasibility of the method in the soft X-ray range. Furthermore, a laser-produced plasma source (LPP) is utilized to investigate the feasibility of this technique in the laboratory. The results from the BESSY II measurements are in good agreement with the simulations and prove the applicability of scan-free GEXRF in the soft X-ray range for quality control and process engineering of 2D nanostructures. The LPP results illustrate the chances and challenges concerning a transfer of the methodology to the laboratory.

Enabling Coarse X-ray Fluorescence Imaging Scans with Enlarged Synchrotron Beam by Means of Mosaic Crystal Defocusing Optics.

Trace elements, functionalized nanoparticles and labeled entities can be localized with sub-mm spatial resolution by X-ray fluorescence imaging (XFI). Here, small animals are raster scanned with a pencil-like synchrotron beam of high energy and low divergence and the X-ray fluorescence is recorded with an energy-dispersive detector. The ability to first perform coarse scans to identify regions of interest, followed by a close-up with a sub-mm X-ray beam is desirable, because overall measurement time and X-ray dose absorbed by the (biological) specimen can thus be minimized. However, the size of X-ray beams at synchrotron beamlines is usually strongly dependent on the actual beamline setup and can only be adapted within specific pre-defined limits. Especially, large synchrotron beams are non-trivial to generate. Here, we present the concept of graphite-based, convex reflection optics for the one-dimensional enlargement of a 1 mm wide synchrotron beam by a factor of 5 to 10 within a 1 m distance. Four different optics are tested and characterized and their reflection properties compared to ray tracing simulations. The general shape and size of the measured reflection profiles agree with expectations. Enhancements with respect to homogeneity and efficiency can be expected with improved optics manufacturing. A mouse phantom is used for a proof-of-principle XFI experiment demonstrating the applicability of coarse and fine scans with the suggested optics design.

Toroidal multilayer mirrors for laboratory soft X-ray grazing emission X-ray fluorescence.

Efficient soft X-ray spectroscopy in the laboratory is still a challenging task. Here, we report on new toroidal multilayer optics designed and applied with the laser-produced plasma (LPP) source of the Berlin Laboratory for innovative X-ray technologies. The optics are described and characterized, and the application of the updated source to scanning-free grazing emission X-ray fluorescence is demonstrated on thermoelectric gold-doped copper oxide nanofilms. The comparison with synchrotron measurements allows estimating a flux on the sample of approximately 7.5 × 109 photons/s in the 1 keV range on a 100 µm × 100 µm spot, emphasizing the suitability of the updated LPP source for the application in photon hungry experiments.

The effect of post-deposition annealing conditions on structural and thermoelectric properties of sputtered copper oxide films.

The development of thin-film thermoelectric applications in sensing and energy harvesting can benefit largely from suitable deposition methods for earth-abundant materials. In this study, p-type copper oxide thin films have been prepared on soda lime silicate glass by direct current (DC) magnetron sputtering at room temperature from a pure copper metallic target in an argon atmosphere, followed by subsequent annealing steps at 300 °C under various atmospheres, namely air (CuO:air), nitrogen (CuO:N) and oxygen (CuO:O). The resultant films have been studied to understand the influence of various annealing atmospheres on the structural, spectroscopic and thermoelectric properties. X-ray diffraction (XRD) patterns of the films showed reflexes that could be assigned to those of crystalline CuO with a thin mixed Cu(I)Cu(II) oxide, which was also observed by near edge X-ray absorption fine structure spectroscopy (NEXAFS). The positive Seebeck coefficient (S) reached values of up to 204 µV K-1, confirming the p-type behavior of the films. Annealing under oxygen provided a significant improvement in the electrical conductivity up to 50 S m-1, resulting in a power factor of 2 µW m-1 K-2. The results reveal the interplay between the intrinsic composition and the thermoelectric performance of mixed copper oxide thin films, which can be finely adjusted by simply varying the annealing atmosphere.

High average power, highly brilliant laser-produced plasma source for soft X-ray spectroscopy.

In this work, a novel laser-produced plasma source is presented which delivers pulsed broadband soft X-radiation in the range between 100 and 1200 eV. The source was designed in view of long operating hours, high stability, and cost effectiveness. It relies on a rotating and translating metal target and achieves high stability through an on-line monitoring device using a four quadrant extreme ultraviolet diode in a pinhole camera arrangement. The source can be operated with three different laser pulse durations and various target materials and is equipped with two beamlines for simultaneous experiments. Characterization measurements are presented with special emphasis on the source position and emission stability of the source. As a first application, a near edge X-ray absorption fine structure measurement on a thin polyimide foil shows the potential of the source for soft X-ray spectroscopy.

Does the exposure mode to ENPs influence their toxicity to aquatic species? A case study with TiO2 nanoparticles and Daphnia magna.

Recent studies suggest that the ecotoxicity of engineered nanoparticles (ENPs) is dependent upon the treatment of ENPs in suspensions (e.g. sonication or use of solvents) and on the mode of exposure to test organisms. We conducted several bioassays with Daphnia magna in order to determine how adverse effects of TiO2 nanoparticles (n-TiO2) are influenced by experimental set-up. Several treatments were applied, including three test media, several treatments of n-TiO2 suspensions (stirring, sonication) and different exposure modes (exposure duration and volume of test suspension). No adverse effects were observed when D. magna were exposed to 50 mL of suspension, regardless of TiO2 concentration (up to 250 mg/L) and exposure duration. Conversely, adverse effects were observed when D. magna were exposed to 2 mL of suspension for 96 h with a 50 % effect concentration EC50 values ranging from 32 mg/L to 82 mg/L. Test media had no significant influence on the outcome of all treatments. For a better mechanistic understanding of the experimental set-up at which adverse effects were observed, the particle size of n-TiO2 in the test media was characterized throughout the test duration. These measurements revealed a fast and strong agglomeration with a secondary particle size in the order of magnitude of micrometers. Our study describes how the effects of n-TiO2 on D .magna are influenced by the duration of exposure and volume of media, highlighting the need for standardization of experimental methods.

The coating makes the difference: acute effects of iron oxide nanoparticles on Daphnia magna.

The surface of nanoparticles (NP) is often functionalized with a capping agent to increase their colloidal stability. Having a strong effect on the characteristics of NP, the coating might already determine the risk from NP to organisms and the environment. In this study identical iron oxide nanoparticles (IONP; Ø 5-6nm) were functionalized with four different coatings: ascorbate (ASC-IONP), citrate (CIT-IONP), dextran (DEX-IONP), and polyvinylpyrrolidone (PVP-IONP). Ascorbate and citrate stabilize NP via electrostatic repulsion whereas dextran and polyvinylpyrrolidone are steric stabilizers. All IONP were colloidally stable over several weeks. Their acute effects on neonates of the waterflea Daphnia magna were investigated over 96h. The highest immobilizing effect was found for ASC- and DEX-IONP. In the presence of neonates, both agglomerated or flocculated and adsorbed to the carapace and filtering apparatuses, inducing high immobilization. Lower immobilization was found for CIT-IONP. Their effect was hypothesized to partly originate from an increased release of dissolved iron and the ability to form reactive oxygen species (ROS). Furthermore, incomplete ecdysis occurred at high concentrations of ASC-, DEX-, and CIT-IONP. PVP-IONP did not induce any negative effect, although high quantities were visibly ingested by the daphnids. PVP-IONP had the highest colloidal stability without any occurring agglomeration, adsorption, or dissolution. Only strong swelling of the PVP coating was observed in medium, highly increasing the hydrodynamic diameter. Each coating caused individual effects. Toxicity cannot be correlated to hydrodynamic diameter or the kind of stabilizing forces. Effects are rather linked to decreasing colloidal stability, the release of ions from the core material or the ability to form ROS, respectively.

Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials.

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K2Cr2O7) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO3). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K2Cr2O7 and AgNPs continued to increase within the prolonged test span. The test comparisons with K2Cr2O7 did not reveal any significant differences between ST and MT. AgNO3 toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.

Intrinsically green iron oxide nanoparticles? From synthesis via (eco-)toxicology to scenario modelling.

Iron oxide nanoparticles (IONP) are currently being studied as green magnet resonance imaging (MRI) contrast agents. They are also used in huge quantities for environmental remediation and water treatment purposes, although very little is known on the consequences of such applications for organisms and ecosystems. In order to address these questions, we synthesised polyvinylpyrrolidone-coated IONP, characterised the particle dispersion in various media and investigated the consequences of an IONP exposure using an array of biochemical and biological assays. Several theoretical approaches complemented the measurements. In aqueous dispersion IONP had an average hydrodynamic diameter of 25 nm and were stable over six days in most test media, which could also be predicted by stability modelling. The particles were tested in concentrations of up to 100 mg Fe per L. The activity of the enzymes glutathione reductase and acetylcholine esterase was not affected, nor were proliferation, morphology or vitality of mammalian OLN-93 cells although exposure of the cells to 100 mg Fe per L increased the cellular iron content substantially. Only at this concentration, acute toxicity tests with the freshwater flea Daphnia magna revealed slightly, yet insignificantly increased mortality. Two fundamentally different bacterial assays, anaerobic activated sludge bacteria inhibition and a modified sediment contact test with Arthrobacter globiformis, both rendered results contrary to the other assays: at the lowest test concentration (1 mg Fe per L), IONP caused a pronounced inhibition whereas higher concentrations were not effective or even stimulating. Preliminary and prospective risk assessment was exemplified by comparing the application of IONP with gadolinium-based nanoparticles as MRI contrast agents. Predicted environmental concentrations were modelled in two different scenarios, showing that IONP could reduce the environmental exposure of toxic Gd-based particles by more than 50%. Application of the Swiss "Precautionary Matrix for Synthetic Nanomaterials" rendered a low precautionary need for using our IONP as MRI agents and a higher one when using them for remediation or water treatment. Since IONP and (considerably more reactive) zerovalent iron nanoparticles are being used in huge quantities for environmental remediation purposes, it has to be ascertained that these particles pose no risk to either human health or to the environment.