Optimization of Mass Spectrometry Imaging for Drug Metabolism and Distribution Studies in the Zebrafish Larvae Model: A Case Study with the Opioid Antagonist Naloxone

Zebrafish (ZF; Danio rerio) larvae have emerged as a promising in vivo model in drug metabolism studies. Here, we set out to ready this model for integrated mass spectrometry imaging (MSI) to comprehensively study the spatial distribution of drugs and their metabolites inside ZF larvae. In our pilot study with the overall goal to improve MSI protocols for ZF larvae, we investigated the metabolism of the opioid antagonist naloxone. We confirmed that the metabolic modification of naloxone is in high accordance with metabolites detected in HepaRG cells, human biosamples, and other in vivo models. In particular, all three major human metabolites were detected at high abundance in the ZF larvae model. Next, the in vivo distribution of naloxone was investigated in three body sections of ZF larvae using LC-HRMS/MS showing that the opioid antagonist is mainly present in the head and body sections, as suspected from published human pharmacological data. Having optimized sample preparation procedures for MSI (i.e., embedding layer composition, cryosectioning, and matrix composition and spraying), we were able to record MS images of naloxone and its metabolites in ZF larvae, providing highly informative distributional images. In conclusion, we demonstrate that all major ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters, as part of in vivo pharmacokinetic studies, can be assessed in a simple and cost-effective ZF larvae model. Our established protocols for ZF larvae using naloxone are broadly applicable, particularly for MSI sample preparation, to various types of compounds, and they will help to predict and understand human metabolism and pharmacokinetics

Drug Administration Routes Impact the Metabolism of a Synthetic Cannabinoid in the Zebrafish Larvae Model

Zebrafish (Danio rerio) larvae have gained attention as a valid model to study in vivo drug metabolism and to predict human metabolism. The microinjection of compounds, oligonucleotides, or pathogens into zebrafish embryos at an early developmental stage is a well-established technique. Here, we investigated the metabolism of zebrafish larvae after microinjection of methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (70N-5F-ADB) as a representative of recently introduced synthetic cannabinoids. Results were compared to human urine data and data from the in vitro HepaRG model and the metabolic pathway of 70N-5F-ADB were reconstructed. Out of 27 metabolites detected in human urine samples, 19 and 15 metabolites were present in zebrafish larvae and HepaRG cells, respectively. The route of administration to zebrafish larvae had a major impact and we found a high number of metabolites when 70N-5F-ADB was microinjected into the caudal vein, heart ventricle, or hindbrain. We further studied the spatial distribution of the parent compound and its metabolites by mass spectrometry imaging (MSI) of treated zebrafish larvae to demonstrate the discrepancy in metabolite profiles among larvae exposed through different administration routes. In conclusion, zebrafish larvae represent a superb model for studying drug metabolism, and when combined with MSI, the optimal administration route can be determined based on in vivo drug distribution

Effects of broccoli extract and various essential oils on intestinal and faecal microflora and on xenobiotic enzymes and the antioxidant system of piglets

Objective: Since the ban of antibiotics as growth promoting feed additives in the EU in 2006 research in alternatives has gained importance. Phytogenic feed additives represent a heterogenous class of different plant derived substances that are discussed to improve the health of farm animals by direct and indirect antioxidant effects and by influencing microbial eubiosis in the gastrointestinal tract. Consequently our study aimed to investigate the influence of broccoli extract and the essential oils of tur- meric, oregano, thyme and rosemary, as selected individual additives, on intestinal and faecal microflora, on xenobiotic enzymes, and on the antioxidant system of piglets. Methods: 48 four weeks old male weaned piglets were assigned to 6 groups of 8. The piglets were housed individually in stainless steel pens with slatted floor. The control group (Con) was fed a diet without an additive for 4 weeks. The diet of group BE contained 0.15 g/kg sulforaphane in form of a broccoli extract. 535, 282, 373 and 476 mg/kg of the essential oils of turmeric (Cuo), oregano (Oo), thyme (To) and rosemary (Ro) were added to the diets of the remaining 4 groups to stan-dardise supplementation to 150 mg/kg of the oils’ key terpene compounds ar-turmerone, carvacrol, thymol and 1,8-cineole. The composition of bacterial microflora was examined by cultivating samples of jejeunal and colonic mucosa and of faeces under specific conditions. The mRNA expression of xenobiotic and antioxidant enzymes was determined by reversing transcrip- tase real time detection PCR (RT-PCR). Total antioxidant status was assayed using the Trolox Equivalent Antioxidant Capacity (TEAC), and lipid peroxidation was determined by measuring thiobarbioturic acid reactive substances (TBA- RS). Results: Compared to Con piglets all additives positively influenced weight gain and feed conversion in week 1. Over the whole trial period no significant differences in performance parameters existed between the experimental groups. Compared to group Con performance of Ro piglets was, however, slightly impaired. Com- pared to Con piglets Cuo, Oo and To increased the ratio of Lactobacilli:E. coli attached to the jejunal mucosa, whereas BE and Ro impaired this ratio slightly. In contrast in colonic mucosa Ro improved Lactobacilli:E. coli ratio. In faecal samples an improvement of Lactobacilli:E. coli ratio could be analysed for To and Ro. Ro was the only additive that reduced the incidence rate of piglets tested positive for enterotoxic E. coli (ETEC). All additives significantly increased jejunal TEAC and reduced TBA-RS. In the liver BE, Cuo, Oo and To increased TEAC in tendency and Ro significantly. Liver TBA-RS were slightly reduced by all additives compared to Con piglets. Whereas the influence of BE, To and Ro on jejunal TEAC mainly was derived from the induction of xenobiotic and antioxidant enzymes (indirect antioxidant effects), Cuo and Oo influenced TEAC by direct antioxidant effects. Discussion and Conclusions: Our results have shown: That within the labiatae oils Oo and To have the potential to improve performance slightly. That phytogenic substances have a small but not sig- nificant influence on intestinal microflora. That phytogenic feed additives up-regulate the anti- oxidant system of piglets either by direct or by indirect antioxidant effects and that they may thereby improve health status. That within the labiatae oils Oo has a high direct antioxidant potential whereas Ro potently induces xenobiotic and antioxidant enzymes. That broccoli extract is an attractive new phytogenic additive, improving antioxidant status by indirect antioxidant effects. That defined combinations of selected phytogenic substances may produce additive effects. That health promoting effects of phytogenic additives in the future should be studied systematically under the challenge with pathogenic microorganisms or food derived to-xins

Chemical ozone loss in the Arctic winter 1991–1992

Chemical ozone loss in winter 1991–1992 is recalculated based on observations of the HALOE satellite instrument, Version 19, ER-2 aircraft measurements and balloon data. HALOE satellite observations are shown to be reliable in the lower stratosphere below 400 K, at altitudes where the measurements are most likely disturbed by the enhanced sulfate aerosol loading, as a result of the Mt.~Pinatubo eruption in June 1991. Significant chemical ozone loss (13–17 DU) is observed below 380 K from Kiruna balloon observations and HALOE satellite data between December 1991 and March 1992. For the two winters after the Mt. Pinatubo eruption, HALOE satellite observations show a stronger extent of chemical ozone loss towards lower altitudes compared to other Arctic winters between 1991 and 2003. In spite of already occurring deactivation of chlorine in March 1992, MIPAS-B and LPMA balloon observations indicate that chlorine was still activated at lower altitudes, consistent with observed chemical ozone loss occurring between February and March and April. Large chemical ozone loss of more than 70 DU in the Arctic winter 1991–1992 as calculated in earlier studies is corroborated here

Magnetoresistance of a two-dimensional electron gas with spatially periodic lateral modulations: Exact consequences of Boltzmann's equation

On the basis of Boltzmann's equation, and including anisotropic scattering in the collision operator, we investigate the effect of one-dimensional superlattices on two-dimensional electron systems. In addition to superlattices defined by static electric and magnetic fields, we consider mobility superlattices describing a spatially modulated density of scattering centers. We prove that magnetic and electric superlattices in $x$-direction affect only the resistivity component $\rho_{xx}$ if the mobility is homogeneous, whereas a mobility lattice in $x$-direction in the absence of electric and magnetic modulations affects only $\rho_{yy}$. Solving Boltzmann's equation numerically, we calculate the positive magnetoresistance in weak magnetic fields and the Weiss oscillations in stronger fields within a unified approach.Comment: submitted to PR

Induction of Liver Size Reduction in Zebrafish Larvae by the Emerging Synthetic Cannabinoid 4F-MDMB-BINACA and Its Impact on Drug Metabolism

Zebrafish (ZF; Danio rerio) larvae have become a popular in vivo model in drug metabolism studies. Here, we investigated the metabolism of methyl 2-[1-(4-fluorobutyl)-1H-indazole-3-carboxamido]- 3,3-dimethylbutanoate (4F-MDMB-BINACA) in ZF larvae after direct administration of the cannabinoid via microinjection, and we visualized the spatial distributions of the parent compound and its metabolites by mass spectrometry imaging (MSI). Furthermore, using genetically modified ZF larvae, the role of cannabinoid receptor type 1 (CB1) and type 2 (CB2) on drug metabolism was studied. Receptor-deficient ZF mutant larvae were created using morpholino oligonucleotides (MOs), and CB2-deficiency had a critical impact on liver development of ZF larva, leading to a significant reduction of liver size. A similar phenotype was observed when treating wild-type ZF larvae with 4F-MDMB-BINACA. Thus, we reasoned that the cannabinoid-induced impaired liver development might also influence its metabolic function. Studying the metabolism of two synthetic cannabinoids, 4F-MDMB-BINACA and methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3- dimethylbutanoate (70N-5F-ADB), revealed important insights into the in vivo metabolism of these compounds and the role of cannabinoid receptor binding

Noseleaf Dynamics during Pulse Emission in Horseshoe Bats

Horseshoe bats emit their biosonar pulses nasally and diffract the outgoing ultrasonic waves by conspicuous structures that surrounded the nostrils. Here, we report quantitative experimental data on the motion of a prominent component of these structures, the anterior leaf, using synchronized laser Doppler vibrometry and acoustic recordings in the greater horseshoe bat (Rhinolophus ferrumequinum). The vibrometry data has demonstrated non-random motion patterns in the anterior leaf. In these patterns, the outer rim of the walls of the anterior leaf twitches forward and inwards to decrease the aperture of the noseleaf and increase the curvature of its surfaces. Noseleaf displacements were correlated with the emitted ultrasonic pulses. After their onset, the inward displacements increased monotonically towards their maximum value which was always reached within the duration of the biosonar pulse, typically towards its end. In other words, the anterior leaf’s surfaces were moving inwards during most of the pulse. Non-random motions were not present in all recorded pulse trains, but could apparently be switched on or off. Such switches happened between sequences of consecutive pulses but were never observed between individual pulses within a sequence. The amplitudes of the emitted biosonar pulse and accompanying noseleaf movement were not correlated in the analyzed data set. The measured velocities of the noseleaf surface were too small to induce Doppler shifts of a magnitude with a likely significance. However, the displacement amplitudes were significant in comparison with the overall size of the anterior leaf and the sound wavelengths. These results indicate the possibility that horseshoe bats use dynamic sensing principles on the emission side of their biosonar system. Given the already available evidence that such mechanisms exist for biosonar reception, it may be hypothesized that time-variant mechanisms play a pervasive role in the biosonar sensing of horseshoe bats

How to Study the Metabolism of New Psychoactive Substances for the Purpose of Toxicological Screenings — A Follow-Up Study Comparing Pooled Human Liver S9, HepaRG Cells, and Zebrafish Larvae

The new psychoactive substances (NPS) market continues to be very dynamic. A large number of compounds belonging to diverse chemical groups continue to emerge. This makes their detection in biological samples challenging for clinical and forensic toxicologists. Knowledge of the metabolic fate of NPS is crucial for developing comprehensive screening procedures. As human studies are not feasible due to ethical concerns, the current study aimed to compare the NPS' metabolic pattern in incubations with pooled human liver S9 fraction (pHLS9), human liver HepaRG cells, and zebrafish larvae. The latter model was recently shown to be a promising preclinical surrogate for human hepatic metabolism of a synthetic cannabinoid. However, studies concerning other NPS classes are still missing and therefore an amphetamine-based N-methoxybenzyl (NBOMe) compound, a synthetic cathinone, a pyrrolidinophenone analog, a lysergamide, as well as another synthetic cannabinoid were included in the current study. Liquid chromatography coupled to Orbitrap-based high-resolution tandem mass spectrometry was used to analyze metabolic data. Zebrafish larvae were found to produce the highest number of phase I but also phase II metabolites (79 metabolites in total), followed by HepaRG cells (66 metabolites). Incubations with pHLS9 produced the least metabolites (57 metabolites). Furthermore, the involvement of monooxygenases and esterases in the metabolic phase I transformations of 4F-MDMB-BINACA was elucidated using single-enzyme incubations. Several cytochrome P450 (CYP) isozymes were shown to contribute, and CYP3A5 was involved in all CYP-catalyzed reactions, while amide and ester hydrolysis were catalyzed by the human carboxylesterase (hCES) isoforms hCES1b and/or hCES1c. Finally, metabolites were compared to those present in human biosamples if data were available. Overall, the metabolic patterns in HepaRG cells provided the worst overlap with that in human biosamples. Zebrafish larvae experiments agreed best with data found in human plasma and urine analysis. The current study underlines the potential of zebrafish larvae as a tool for elucidating the toxicokinetics of NPS in the future

Long-range transport pathways of tropospheric source gases originating in Asia into the northern lower stratosphere during the Asian monsoon season 2012

Global simulations with the Chemical Lagrangian Model of the Stratosphere (CLaMS) using artificial tracers of air mass origin are used to analyze transport mechanisms from the Asian monsoon region into the lower stratosphere. In a case study, the transport of air masses from the Asian monsoon anticyclone originating in India/China by an eastward-migrating anticyclone which broke off from the main anticyclone on 20 September 2012 and filaments separated at the northeastern flank of the anticyclone are analyzed. Enhanced contributions of young air masses (younger than 5 months) are found within the separated anticyclone confined at the top by the thermal tropopause. Further, these air masses are confined by the anticyclonic circulation and, on the polar side, by the subtropical jet such that the vertical structure resembles a bubble within the upper troposphere. Subsequently, these air masses are transported eastwards along the subtropical jet and enter the lower stratosphere by quasi-horizontal transport in a region of double tropopauses most likely associated with Rossby wave breaking events. As a result, thin filaments with enhanced signatures of tropospheric trace gases were measured in the lower stratosphere over Europe during the TACTS/ESMVal campaign in September 2012 in very good agreement with CLaMS simulations. Our simulations demonstrate that source regions in Asia and in the Pacific Ocean have a significant impact on the chemical composition of the lower stratosphere of the Northern Hemisphere. Young, moist air masses, in particular at the end of the monsoon season in September/October 2012, flooded the extratropical lower stratosphere in the Northern Hemisphere with contributions of up to ≈30% at 380K (with the remaining fraction being aged air). In contrast, the contribution of young air masses to the Southern Hemisphere is much lower. At the end of October 2012, approximately 1.5 ppmv H$_{2}$O is found in the lower Northern Hemisphere stratosphere (at 380 K) from source regions both in Asia and in the tropical Pacific compared to a mean water vapor content of ≈5 ppmv. In addition to this main transport pathway from the Asian monsoon anticyclone to the east along the subtropical jet and subsequent transport into the northern lower stratosphere, a second horizontal transport pathway out of the anticyclone to the west into the tropics (TTL) is found in agreement with MIPAS HCFC-22 measurements

Evaluation of extraction methods for untargeted metabolomic studies for future applications in zebrafish larvae infection models

Sample preparation in untargeted metabolomics should allow reproducible extractions of as many molecules as possible. Thus, optimizing sample preparation is crucial. This study compared six diferent extraction procedures to fnd the most suitable for extracting zebrafsh larvae in the context of an infection model. Two one-phase extractions employing methanol (I) and a single miscible phase of methanol/acetonitrile/water (II) and two two-phase methods using phase separation between chloroform and methanol/water combinations (III and IV) were tested. Additional bead homogenization was used for methods III and IV (III_B and IV_B). Nine internal standards and 59 molecules of interest (MoInt) related to mycobacterial infection were used for method evaluation. Two-phase methods (III and IV) led to a lower feature count, higher peak areas of MoInt, especially amino acids, and higher coefcients of variation in comparison to one-phase extractions. Adding bead homogenization increased feature count, peak areas, and CVs. Extraction I showed higher peak areas and lower CVs than extraction II, thus being the most suited one-phase method. Extraction III and IV showed similar results, with III being easier to execute and less prone to imprecisions. Thus, for future applications in zebrafsh larvae metabolomics and infection models, extractions I and III might be chosen