Long-term effects of a catastrophic insecticide spill on stream invertebrates.

Accidental spills or illegal discharges of pesticides in aquatic ecosystems can lead to exposure levels that strongly exceed authorized pesticide concentrations, causing major impacts on aquatic ecosystems. Such short-term events often remain undetected in regular monitoring programs with infrequent sampling. In early spring 2015, we identified a catastrophic pesticide spill with the insecticide cypermethrin in the Holtemme River, Germany. Based on existing pre-event macroinvertebrate community data, we monitored the effects and recovery of the macroinvertebrate community for more than two years after the spill. Strong short-term effects were apparent for all taxa with the exception of Chironomidae and Tubificidae. Effects could also be observed on the community level as total abundance, taxa number and biomass strongly decreased. Total abundance and taxa number showed a fast recovery. Regarding long-term effects, the total biomass remained substantially below the pre-contamination level (76%) until the end of the study. Also the abundances of three taxa (Gammarus, Leuctra, Limnius Ad.) did not return to levels prior to the spill even after 26 months. This lack of the taxon-specific recovery was likely due to their long generation time and a low migration ability due to a restricted connectivity between the contaminated site and uncontaminated stream sections. These factors proved to be stronger predictors for the recovery than the pesticide tolerance. We revealed that the biological indicators SPEARpesticides and share of Ephemeroptera, Plecoptera and Trichoptera (EPT) are not suitable for the identification of such extreme events, when nearly all taxa are eradicated. Both indicators are functioning only when repeated stressors initiate long-term competitive replacement of sensitive by insensitive taxa. We conclude that pesticide spills can have significant long-term effects on stream macroinvertebrate communities. Regular ecological monitoring is imperative to identify such ecosystem impairments, combined with analytical chemistry methods to identify the potential sources of spills.

Combined Ion-Trapping and Mass Balance Models To Describe the pH-Dependent Uptake and Toxicity of Acidic and Basic Pharmaceuticals in Zebrafish Embryos ( Danio rerio).

The aim of the current study was to understand and develop models to predict the pH-dependent toxicity of ionizable pharmaceuticals in embryos of the zebrafish Danio rerio. We found a higher uptake and toxicity with increasing neutral fraction of acids (diclofenac, genistein, naproxen, torasemide, and warfarin) and bases (metoprolol and propranolol). Simple mass balance models accounting for the partitioning to lipids and proteins in the zebrafish embryo were found to be suitable to predict the bioconcentration after 96 h of exposure if pH values did not differ much from the internal pH of 7.55. For other pH values, a kinetic ion-trap model for the zebrafish embryo explained the pH dependence of biouptake and toxicity. The total internal lethal concentrations killing 50% of the zebrafish embryos (ILC50) were calculated from the measured BCF and LC50. The resulting ILC50 were independent of external pH. Critical membrane concentrations were deduced by an internal mass balance model, and apart from diclofenac, whose specific toxicity in fish had already been established, all pharmaceuticals were confirmed to act as baseline toxicants in zebrafish.

pH-Dependent Uptake and Sublethal Effects of Antihistamines in Zebrafish (Danio rerio) Embryos.

Reported off-target effects of antihistamines in humans draw interest in ecotoxicity testing of first- and second-generation antihistamines, the latter of which have fewer reported side effects in humans. Because antihistamines are ionizable compounds, the pH influences uptake and toxicity and thus is highly relevant when conducting toxicity experiments. Zebrafish embryo toxicity tests were performed with the 3 first-generation antihistamines ketotifen, doxylamine, and dimethindene and the 2 second-generation antihistamines cetirizine and levocabastine at pH 5.5, 7.0, and 8.0. We detected effects on survival, phenotype, swimming activity, and heart rate for 4 antihistamines with the exception of levocabastine, which did not show any lethal or sublethal effects. When compared to lethal concentrations, effect concentrations neither of phenotype malformation nor of swimming activity or heart rate deviated by more than a factor of 10 from lethal concentrations, indicating that all sublethal effects were fairly nonspecific. First-generation antihistamines are weak bases and showed decreasing external effect concentrations with increasing neutral fraction, accompanied by increased uptake in the fish embryo. As a result, internal effect concentrations were independent from external pH. The pH-dependent toxicity originates from speciation-dependent uptake, with neutral species taken up in higher amounts than the corresponding ionic species. Cetirizine, which shifts from a zwitterionic to an anionic state in the measured pH range, did not show any pH-dependent uptake or toxicity. Environ Toxicol Chem 2019;00:1-11. © 2019 SETAC.

Influence of pH on the uptake and toxicity of ß-blockers in embryos of zebrafish, Danio rerio.

ß-Blockers are weak bases with acidity constants related to their secondary amine group. At environmental pH they are protonated with the tendency to shift to their neutral species at more alkaline pH. Here we studied the influence of pH from 5.5 to 8.6 on the toxicity of the four ß-blockers atenolol, metoprolol, labetalol and propranolol in zebrafish embryos, relating toxicity not only in a conventional way to external aqueous concentrations but also to measured internal concentrations. Besides lethality, we evaluated changes in swimming activity and heartbeat, using the Locomotor Response (LMR) method and the Vertebrate Automated Screening Technology (VAST) for high throughput imaging. Effects of metoprolol, labetalol and propranolol were detected on phenotype, heart rate and swimming activity. External effect concentrations decreased with increasing neutral fraction for all three pharmaceuticals, attributed by an enhanced uptake of the neutral species in comparison to the corresponding charged form. The LC50 of metoprolol decreased by a factor of 35 from 1.91 mM with almost complete cationic state at pH 7.0 to 0.054 mM with 8% neutral fraction at pH 8.6. For propranolol the LC50 of 2.42 mM at pH 5.5 was even 100 fold higher than the LC50 at pH 8 with 0.023 mM where 3% were neutral fraction. No effects were detected in the zebrafish embryo exposed to atenolol. The internal concentrations for metoprolol and propranolol were quantified at non-toxic concentrations and at the LC10. Apparent bioconcentration factors (BCF) ranged from 1.96 at pH 7.0 to 32.0 at pH 8.6 for metoprolol and from 1.86 at pH 5.5 to 169 at pH 8.0 for propranolol. The BCFs served to predict the internal effect concentrations from the measured external effect concentrations. Internal effect concentrations of metoprolol and propranolol were in a similar range for all pH-values and for all endpoints. Interestingly, the internal effect concentrations were in the internal concentration range of baseline toxicity, which suggests that the effects of the ß-blockers are rather unspecific, even for sublethal effects on heart rate. In summary, our data confirm that the pH-dependent toxicity related to external concentrations can be explained by toxicokinetic effects and that the internal effect concentrations are pH-independent.

The Copper Efflux Regulator CueR Is Subject to ATP-Dependent Proteolysis in Escherichia coli.

The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes copA and cueO via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that Escherichia coli CueR is prone to proteolysis by the AAA+ proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in E. coli by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.

When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli.

Cellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA+ proteases in Escherichia coli FtsH is the only essential and membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain. FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Recent trapping approaches have revealed a number of novel FtsH substrates. This review summarizes the substrate diversity of FtsH and presents details on the surprisingly diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.

Mini review: ATP-dependent proteases in bacteria.

AAA(+) proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA(+) proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA(+) protease variants. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 505-517, 2016.

Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron.

Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm.