Zebrafish Oatp1d1 Acts as a Cellular Efflux Transporter of the Anionic Herbicide Bromoxynil.

Toxicokinetics (TK) of ionic compounds in the toxico-/pharmacological model zebrafish embryo (Danio rerio) depend on absorption, distribution, metabolism, and elimination (ADME) processes. Previous research indicated involvement of transport proteins in the TK of the anionic pesticide bromoxynil in zebrafish embryos. We here explored the interaction of bromoxynil with the organic anion-transporting polypeptide zebrafish Oatp1d1. Mass spectrometry imaging revealed accumulation of bromoxynil in the gastrointestinal tract of zebrafish embryos, a tissue known to express Oatp1d1. In contrast to the Oatp1d1 reference substrate bromosulfophthalein (BSP), which is actively taken up by transfected HEK293 cells overexpressing zebrafish Oatp1d1, those cells accumulated less bromoxynil than empty vector-transfected control cells. This indicates cellular efflux of bromoxynil by Oatp1d1. This was also seen for diclofenac but not for carbamazepine, examined for comparison. Correspondingly, internal concentrations of bromoxynil and diclofenac in the zebrafish embryo were increased when coexposed with BSP, inhibiting the activities of various transporter proteins, including Oatp1d1. The effect of BSP on accumulation of bromoxynil and diclofenac was enhanced in further advanced embryo stages, indicating increased efflux activity in those stages. An action of Oatp1d1 as an efflux transporter of ionic environmental compounds in zebrafish embryos should be considered in future TK assessments.

Effective processing and evaluation of chemical imaging data with respect to morphological features of the zebrafish embryo.

A workflow was developed and implemented in a software tool for the automated combination of spatially resolved laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) data and data on the morphology of the biological tissue. Making use of a recently published biological annotation software, FishImager automatically assigns the biological feature as regions of interest (ROIs) and overlays them with the quantitative LA-ICP-MS data. Furthermore, statistical tools including cluster algorithms can be applied to the elemental intensity data and directly compared with the ROIs. This is effectively visualized in heatmaps. This allows gaining statistical significance on distribution and co-localization patterns. Finally, the biological functions of the assigned ROIs can then be easily linked with elemental distributions. We demonstrate the versatility of FishImager with quantitative LA-ICP-MS data of the zebrafish embryo tissue. The distribution of natural elements and xenobiotics is analyzed and discussed. With the help of FishImager, it was possible to identify compartments affected by toxicity effects or biological mechanisms to eliminate the xenobiotic. The presented workflow can be used for clinical and ecotoxicological testing, for example. Ultimately, it is a tool to simplify and reproduce interpretations of imaging LA-ICP-MS data in many applications.

Yolk Sac of Zebrafish Embryos as Backpack for Chemicals?

The zebrafish embryo (Danio rerio) has developed into one of the most important nonsentient animal models for the hazard assessments of chemicals, but the processes governing its toxicokinetics (TK) are poorly understood. This study compares the uptake of seven test compounds into the embryonic body and the yolk sac of the zebrafish embryo using TK experiments, a dialysis approach, thermodynamic calculations, and kinetic modeling. Experimental data show that between 95% (4-iodophenol) and 67% (carbamazepine) of the total internal amount in 26 h post fertilization (hpf) embryos and between 80 and 49% in 74 hpf embryos were found in the yolk. Thus, internal concentrations determined for the whole embryo overestimate the internal concentration in the embryonic body: for the compounds of this study, up to a factor of 5. Partition coefficients for the embryonic body and a one-compartment model with diffusive exchange were calculated for the neutral test compounds and agreed reasonably with the experimental data. For prevalently ionic test compounds at exposure pH (bromoxynil, paroxetine), however, the extent and the speed of uptake were low and could not be modeled adequately. A better understanding of the TK of ionizable test compounds is essential to allow assessment of the validity of this organismic test system for ionic test compounds.

Elemental imaging (LA-ICP-MS) of zebrafish embryos to study the toxicokinetics of the acetylcholinesterase inhibitor naled.

The zebrafish embryo is an important model in ecotoxicology but the spatial distribution of chemicals and the relation to observed effects is not well understood. Quantitative imaging can help to gain insights into the distribution of chemicals in the zebrafish embryo. Laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) is used to quantify the uptake and the uptake kinetics of the bromine (Br) containing organophosphate naled (Dibrom®, dimethyl-1,2-dibromo-2,2-dichloroethylphosphate) and its distribution in zebrafish embryos using Br as the marker element. During exposure, the Br amounts increase in the embryos parallel to the irreversible inhibition of the acetylcholinesterase (AChE). The final amount of Br in the embryo (545 pmol/embryo) corresponds to a 280-fold enrichment of naled from the exposure solution. However, LC-MS/MS analyses showed that the internal concentration of naled remained below the LOD (7.8 fmol/embryo); also the concentration of its known transformation product dichlorvos remained low (0.85 to 2.8 pmol/embryo). These findings indicate the high reactivity and high transformation rate of naled to other products than dichlorvos. 12C normalized intensity distributions of Br in the zebrafish embryo showed an enrichment of Br in its head region. Kernel density estimates of the LA-ICP-MS data were calculated and outline the high reproducibility between replicated and the shift in the Br distribution during exposure. The Br enrichment indicates a preferential debromination or direct covalent reaction of naled with AChE in this region. Graphical abstract ᅟ.

Moxidectin toxicity to zebrafish embryos: Bioaccumulation and biomarker responses.

Moxidectin is an antiparasitic drug belonging to the class of the macrocyclic lactones, subgroup mylbemicins. It is used worldwide in veterinary practice, but little is known about its potential environmental risks. Thus, we used the zebrafish embryo as a model system to study the potential effects of moxidectin on aquatic non-target organisms. The analyses were performed in two experimental sets: (1) acute toxicity and apical endpoints were characterized, with biomarker assays providing information on the activity levels of catalase (CAT), glutathione S-transferase (GST), lactate dehydrogenase (LDH), and acetylcholinesterase (AChE); and (2) internal concentration and spatial distribution of moxidectin were determined using ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-QToF-MS) and matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSi). The acute toxicity to zebrafish embryos (96 hpf) appeared mainly as a decrease in hatching rates (EC50 = 20.75 µg/L). It also altered the enzymatic activity of biomarker enzymes related to xenobiotic processing, anaerobic metabolism, and oxidative stress (GST, LDH, and CAT, respectively) and strongly accumulated in the embryos, as internal concentrations were 4 orders of magnitude higher than those detected in exposure solutions. MALDI-MSi revealed accumulations of the drug mainly in the head and eyes of the embryos (72 and 96 hpf). Thus, our results show that exposure to moxidectin decreases hatching success by 96 h and alters biochemical parameters in the early life stages of zebrafish while accumulating in the head and eye regions of the animals, demonstrating the need to prioritize this compound for environmental studies.

Small streams-large concentrations? Pesticide monitoring in small agricultural streams in Germany during dry weather and rainfall.

Few studies have examined the exposure of small streams (< 30 km2 catchment size) to agriculturally used pesticides, compared to large rivers. A total of 105 sites in 103 small agricultural streams were investigated for 76 pesticides (insecticides, herbicides, fungicides) and 32 pesticide metabolites in spring and summer over two years (2018 and 2019) during dry weather and rainfall using event-driven sampling. The median total concentration of the 76 pesticides was 0.18 µg/L, with 9 pesticides per sample on average (n = 815). This is significantly higher than monitoring data for larger streams, reflecting the close proximity to agricultural fields and the limited dilution by non-agricultural waters. The frequency of detection of all pesticides correlated with sales quantity and half-lives in water. Terbuthylazine, MCPA, boscalid, and tebuconazole showed the highest median concentrations. The median of the total concentration of the 32 metabolites exceeded the pesticide concentration by more than an order of magnitude. During dry weather, the median total concentration of the 76 pesticides was 0.07 µg/L, with 5 pesticides per sample on average. Rainfall events increased the median total pesticide concentration by a factor of 10 (to 0.7 µg/L), and the average number of pesticides per sample to 14 (with up to 41 in single samples). The concentration increase was particularly strong for 2,4-D, MCPA, terbuthylazine, and nicosulfuron (75 percentile). Metabolite concentrations were generally less responsive to rainfall, except for those of terbuthylazine, flufenacet, metamitron, and prothioconazole. The frequent and widespread exceedance of the regulatory acceptable concentrations (RAC) of the 76 pesticides during both, dry weather and rainfall, suggests that current plant protection product authorization and risk mitigation methods are not sufficient to protect small streams.

CB2-deficiency is associated with a stronger hypertrophy and remodeling of the right ventricle in a murine model of left pulmonary artery occlusion.

AIMS: Pulmonary hypertension (PH) leads to right ventricular (RV) adaptation and remodeling and has deleterious long-term effects on RV function. The endocannabinoid receptor CB2 has been associated with protective effects in adaptation and remodeling of the left ventricle after ischemia. Therefore, we investigated the role of CB2 receptor in RV adaptation after occlusion of the left pulmonary artery (LPA) in a murine model. MAIN METHODS: C57/Bl6 (WT)- and CB2 receptor-deficient (Cnr2-/-)-mice underwent paramedian sternotomy and LPA was occluded using a metal clip. Right heart hemodynamic study (Millar®) preceded organ harvesting for immunohistochemistry and mRNA analysis 7 and 21 days (d) post-occlusion. KEY FINDINGS: LPA occlusion led to higher RV systolic pressure in Cnr2-/--hearts, while hemodynamics were comparable with WT-hearts after 21d. Cnr2-/--hearts showed higher macrophage infiltration and lower interleukin-10 expression after 7 d, but otherwise a comparable inflammatory mediator expression profile. Cardiomyocyte-hypertrophy was stronger in Cnr2-/--mice, presenting with higher tenascin-C expression than WT-hearts. Planimetry revealed higher collagen area in Cnr2-/--hearts and small areas of cardiomyocyte-loss. Surrounding cardiomyocytes were cleaved caspase-3- and TUNEL positive in Cnr2-/--hearts. This was associated by maladaptation of myosin heavy-chain isoforms and lower reactive oxygen scavenger enzymes induction in Cnr2-/--hearts. We found comparable morphological changes in both lungs between the two genotypes. SIGNIFICANCE: LPA occlusion led to increased systolic pressure and adaptation of RV in CB2-deficient mice. CB2 receptor seems to modulate RV adaptation through expression of contractile elements, reactive oxygen scavenger enzymes, and inflammatory response in order to prevent cardiomyocyte apoptosis.

The presence of mercury and other trace metals in surface soils in the Norwegian Arctic.

Svalbard is an important study area for investigating the long-range transport of mercury (Hg) and other trace elements to the Arctic. Few studies have focused on their concentrations in Arctic soils. With ongoing climate change leading to thawing permafrost ground the soil compartment is of increasing importance in the Arctic. In this study, elemental composition and soil organic matter (SOM) content of surface and mineral soils in Svalbard are presented. The aim is to provide new data on soils in the Arctic and to gain more knowledge about the role of the soil in the biogeochemical cycle of mercury (Hg). Concentrations are reported for Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, S and Zn. Samples were taken in Adventdalen and in the area near Ny-Ålesund. We obtained a mean Hg concentration of 0.111 ± 0.036 µg/g in surface soils (range 0.041-0.254 µg/g). Hg levels in mineral soils (mean: 0.025 ± 0.013 µg/g; range: 0.004-0.060 µg/g) were substantially lower than in the corresponding surface soils. Hg strongly accumulates in the surface soil layer (upper 3 cm) and is associated with SOM (surface soil: 59 ± 14%). Hg concentrations in the surface soil were slightly lower than those in the humus layer in mainland Norway and were comparable to levels in soils elsewhere in the Arctic. An inverse association of Hg was found with elements attributed to the mineral soil, indicating that Hg is predominantly derived from atmospheric deposition.