The Uptake of Sporopollenin Exine Capsules and Associated Bioavailability of Adsorbed Oestradiol in Selected Aquatic Invertebrates.

Lycopodium clavatum sporopollenin exine capsules (SpECs) are known to both adsorb and absorb chemicals. The aim of the present work was to determine whether oestradiol (E2) is 'bioavailable' to bioindicator species, either pre-adsorbed to, or in the presence of, SpECs. SpEC uptake was confirmed for Daphnia magna and Dreissena bugensis. E2 levels varied among treatments for Caenorhabditis elegans though there was no relationship to SpEC load. E2 was not detected in D. bugensis tissues. Expression changes of general stress and E2-specific genes were measured. For C. elegans, NHR-14 expression suggested that SpECs modulate E2 impacts, but not general health responses. For D. magna, SpECs alone and with E2 changed Vtg1 and general stress responses. For D. bugensis, SpECS were taken up but no E2 or change in gene expression was detected after exposure to E2 and/or SpECs. The present study is the first to investigate SpECs and bound chemical dynamics.

Limited effects of pesticides on stream macroinvertebrates, biofilm nematodes, and algae in intensive agricultural landscapes in Sweden.

Pesticides are frequently detected in surface waters, sometimes at levels exceeding ecotoxicological guidelines. We screened for almost 100 pesticides in 32 streams from intense agricultural areas in Southern Sweden, in concert with water chemistry parameters. In addition, we investigated the communities of benthic macroinvertebrates, biofilm nematodes and algae and calculated multiple bioassessment metrics. The number of pesticides found in each stream ranged between 2 and 52, but the sum of Toxic Units (ΣTU) for the mixtures was generally low, and exceeded the European Uniform Principles only in a single sample for algae and in 2% of the samples for Daphnia. Only nematode communities were significantly correlated with the ΣTU, potentially due to their higher pesticide exposure in biofilms. Diatom metrics showed that most streams were impacted by eutrophication and macroinvertebrate metrics showed good status in most streams, whereas the SPEARpesticides (SPEcies At Risk) index, specifically designed to indicate pesticide effects, showed that about half of the samples were at risk. Interestingly, SPEARpesticides was not correlated to ΣTUDaphnia, and this discrepancy suggests that redefining the boundaries for quality classes might be necessary for this index. Moreover, SPEARpesticides was positively correlated with the commonly used macroinvertebrate index ASPT, although disparate results were found for several streams. We argue that this questions the scaling of both metrics and the specificity of their responses. We discuss that the overall good/moderate status of the streams, despite the intense agriculture in the catchments, can be due to the fact that i) a sampling strategy with repeated grab samples did not capture peak pesticide concentrations, thus underestimating acute exposure, ii) pesticide run-off indeed was low, due to measures such as buffer strips, and iii) the nutrient-rich conditions and high sediment loads counteracted pesticide toxicity. We conclude that agricultural land use was the overriding stressor in the investigated streams, including strong effects of nutrients, less apparent effects of pesticides and likely impact of hydromorphological alterations (not specifically addressed in this study).

Pro-Angiogenic Effects of Latent Heparanase and Thrombin Receptor-Mediated Pathways-Do They Share a Common Ground in Melanoma Cells?

Heparanase (HPSE) is an endo-ß-D-glucuronidase that cleaves heparan sulphate (HS) chains of proteoglycans (HSPGs). Besides a remodelling of the extracellular matrix, HPSE increases the bioavailability of pro-angiogenic mediators, such as HS-associated vascular endothelial growth factor (VEGF), thereby contributing to metastatic niche formation. Notably, HPSE also induces release of VEGF from tumour cells independent of its enzymatic activity, but the underlying molecular mechanisms remain unresolved. We found that exogenous addition of latent HPSE stimulates VEGF release from human MV3 melanoma cells. The same effect was noted upon direct stimulation of thrombin receptor (protease-activated receptor 1 [PAR-1]) by Thrombin Receptor Activator Peptide 6 (TRAP-6). The matricellular ligand cysteine-rich 61 protein (Cyr61) was identified as pathway component since Cyr61 knockdown in MV3 cells abolished the VEGF release by TRAP-6 and HPSE. Since both TRAP-6 and HPSE mediated an up-regulation of phosphorylated focal adhesion kinase, which could be blocked by antagonizing PAR-1, we postulated a crosstalk between latent HPSE and PAR-1 in promoting pro-angiogenic pathways. To test this hypothesis at a molecular level, we applied dynamic mass redistribution (DMR) technique measuring intracellular mass relocation as consequence of direct receptor activation. Indeed, latent HPSE evoked a concentration-dependent DMR signal in MV3 cells as TRAP-6 did. Both could be modulated by targeting G-protein receptor signalling in general or by the PAR-1 inhibitor RWJ 56110. Using cells devoid of cell surface HS synthesis, we could confirm HPSE effects on PAR-1, independent of HSPG involvement. These data indicate, for the first time, a crosstalk between latent HPSE, thrombin receptor activation and G-protein signalling in general.

Mass-Sensitive Biosensor Systems to Determine the Membrane Interaction of Analytes.

Biosensors are devices that transform a biological interaction into a readout signal, which is evaluable for analytical purposes. The general strength of biosensor approaches is the avoidance of time-consuming and cost-intensive labeling procedures of the analytes. In this chapter, we give insight into a mass-sensitive surface-acoustic wave (SAW) biosensor, which represents an elegant and highly sensitive method to investigate binding events at a molecular level. The principle of SAW technology is based on the piezoelectric properties of the sensors, so as to binding events and their accompanied mass increase at the sensor surface are detectable by a change in the oscillation of the surface acoustic wave. In combination with model membranes, transferred to the sensor surface, the analytical value of SAW biosensors has strongly been increased and extended to different topics of biomedical investigations, including antibiotic research. The interaction with the bacterial membrane or certain target structures therein is the essential mode of action for various antibacterial compounds. Beside targeted interaction, an unspecific membrane binding or membrane insertion of drugs can contribute to the antibacterial activity by changing the lateral order of membrane constituents or by interfering with the membrane barrier function. Those pleiotropic effects are hardly to illustrate in the bacterial systems and need a detailed view at the in vitro level. Here, we illustrate the usefulness of a SAW biosensor in combination with model membranes to investigate the mode of membrane interaction of antibiotic active peptides. Using two different peptides we exemplary describe the interaction analysis in a two-step gain of information: (1) a binding intensity or affinity by analyzing the phase changes of oscillation, and (2) mode of membrane interaction, i.e., surface binding or internalization of the peptide by following the amplitude of oscillation.

A comparative approach using ecotoxicological methods from single-species bioassays to model ecosystems.

Soft sediments are often hotspots of chemical contamination, and a thorough ecotoxicological assessment of this habitat can help to identify the causes of stress and to improve the health of the respective ecosystems. As an important component of the ecologically relevant meiobenthic fauna, nematodes can be used for sediment assessments, with various assay tools ranging from single-species toxicity tests to field studies. In the present study, microcosms containing sediment were used to investigate direct and indirect effects of zinc on natural nematode assemblages, and acute community toxicity tests considering only direct toxicity were conducted. The responses of the various freshwater nematode species in both approaches were compared with those of Caenorhabditis elegans, determined in standardized tests (ISO 10872). At a median lethal concentration (LC50) of 20 mg Zn/L, C. elegans represented the median susceptibility of 15 examined nematode species examined in the acute community toxicity tests. In the microcosms, Zn affected the nematodes dose-dependently, with changes in species composition first detected at 13 mg Zn/kg to 19 mg Zn/kg sediment dry weight. The observed species sensitivities in the microcosms corresponded better to field observations than to the results of the acute community toxicity tests. Environ Toxicol Chem 2016;35:2987-2997. © 2016 SETAC.

Response of bacteria and meiofauna to iron oxide colloids in sediments of freshwater microcosms.

The use of colloidal iron oxide (FeOx) in the bioremediation of groundwater contamination implies its increasing release into the environment and requires an assessment of its ecotoxicological risk. Therefore, microcosm experiments were carried out to investigate the impact of ferrihydrite colloids on the bacterial and meiofaunal communities of pristine freshwater sediments. The effects of ferrihydrite colloids were compared with those of ferrihydrite macroaggregates to discriminate between colloid-specific and general FeOx impacts. The influence of ferrihydrite colloids on the toxicity of sediment-bound fluoranthene was also considered. At high concentrations (496 mg Fe kg(-1) sediment dry wt), ferrihydrite colloids had a significant, but transient impact on bacterial and meiofaunal communities. Although bacterial community composition specifically responded to ferrihydrite colloids, a more general FeOx effect was observed for meiofauna. Bacterial activity responded most sensitively (already at 55 mg Fe kg(-1) dry wt) without the potential of recovery. Ferrihydrite colloids did not influence the toxicity of sediment-bound fluoranthene. Significant correlations between bacterial activity and meiofaunal abundances were indicative of trophic interactions between bacteria and meiofauna and therefore of the contribution of indirect food web effects to the observed impacts. The results suggest that the application of ferrihydrite colloids for remediation purposes in the field poses no risk for benthic communities, given that, with the exception of generic bacterial activity, any negative effects on communities were reversible.

Latent heparanase facilitates VLA-4-mediated melanoma cell binding and emerges as a relevant target of heparin in the interference with metastatic progression.

Heparanase is an endo-ß-glucuronidase that enzymatically cleaves heparan sulfates (HS) and heparan sulfate proteoglycan (HSPG) structures. Heparanase expression levels by tumors were correlated with cell invasion, angiogenic activity, and poor prognosis. Heparanase can also possess pro-tumorigenic effects independent of its enzymatic activity. Using human melanoma MV3 cells, we demonstrate that latent heparanase activates in a tightly temporary-regulated manner the binding function of the integrin very late antigen-4 (VLA-4), an important component in the metastatic spread of melanoma cells. shRNA-mediated knockdown of syndecan-4 (SDC-4) indicated that this proteoglycan is the key element to convey heparanase binding via focal adhesion complex formation, detected by vinculin staining, to an upregulated VLA-4 binding function. This inside-out signaling pathway of VLA-4 involved activated FAK and Akt, but apparently not PKCα/δ. VLA-4, however, appears representative of other integrins which together impact the heparanase/integrin activation axis in tumorigenicity. Biosensor measurements provided an insight as to how heparin can interfere with this activation process. While low-molecular-weight heparin (LMWH) cannot replace heparanase bound to SDC-4, LMWH can compete with SDC-4 binding of heparanase. Since blockade of heparanase by LMWH has functional consequences for reduced VLA-4 binding, latent heparanase appears as a novel, so far unnoticed target of heparin, underlying its antimetastatic activity.

Using meiofauna to assess pollutants in freshwater sediments: a microcosm study with cadmium.

The direct and indirect effects of Cd on benthic communities were assessed in a freshwater microcosm study over a period of seven months (218 d). Cadmium was regarded as a model substance to evaluate the usefulness of small-scale laboratory microcosm with microscopic fauna. In particular, effects on the meiofauna community, an ecologically important but rather neglected benthic component, were investigated. In addition, some microfaunal parameters (protozoan abundance and microbial activity) were determined. The sediment was spiked with nominal Cd concentrations of 10, 100, and 1,000 mg/kg dry weight. Because of the strong binding of Cd to sediment particles, measured Cd pore-water concentrations never exceeded 129.5 ± 40.7 µg/L. At 1,000 mg/kg dry weight, the abundances of the two dominant meiofauna taxa, nematodes and oligochaetes, were significantly reduced throughout the present study. Regarding nematodes, species of bacterivorous taxa (Daptonema, Eumonhystera) decreased, whereas species of predacious and omnivorous taxa (Mononchus, Dorylaimus, and Ironus) increased in dominance in microcosms of the highest Cd concentration. Transient effects on microfauna were observed, especially in the first half of the present study, with a reduction in microbial activity and protozoan abundance. However, in microcosms receiving the highest Cd concentration, the abundance of the flagellate Euglena mutabilis increased significantly toward the end of the present study. The results of the present study support the use of small-scale microcosms with natural meiofauna communities as a suitable tool to assess the impact of pollutants in freshwater sediments.

Hormonelike effects of humic substances on fish, amphibians, and invertebrates.

Humic substances comprise the majority of organic matter in freshwater ecosystems and were thought to be inert or refractory, except for photolytic degradation. However, evidence is increasing that humic substances interact with aquatic organisms similarly to weak anthropogenic chemicals with nonspecific and specific effects. One specific effect is a hormonelike effect, namely, modulation of the number of offspring, which was first described with the nematode Caenorhabditis elegans. Yet a hormonelike effect is not restricted to only the nematode. With the ornamental swordtail fish, Xiphophorus helleri, and the South African clawed frog, Xenopus laevis, we present phenomenological evidence that slight feminization occurred when these vertebrate species were exposed to a synthetic humic substance, a condensation product of polyphenols. The slight feminization was dose dependent.