Pharmacological modulation of fish-induced depth selection in D. magna: the role of cholinergic and GABAergic signalling.

Animal behaviour is closely related to individual fitness, which allows animals to choose suitable mates or avoid predation. The central nervous system regulates many aspects of animal behaviour responses. Therefore, behavioural responses can be especially sensitive to compounds with a neurodevelopmental or neurofunctional mode of action. Phototactic behavioural changes against fish in the freshwater crustacean Daphnia magna have been the subject of many ecological investigations. The aim of this study was to identify which neurotransmitter systems modulate phototactic behaviour to fish kairomones. We used a positive phototactic D. magna clone (P132,85) that shows marked negative phototactism after exposure to fish kairomones. Treatments included up to 16 known agonists and antagonists of the serotonergic, cholinergic, dopaminergic, histaminergic, glutamatergic and GABAergic systems. It was hypothesized that many neurological signalling pathways may modulate D. magna phototactic behaviour to fish kairomones. A new custom-designed device with vertically oriented chambers was used, and changes in the preferred areas (bottom, middle, and upper areas) were analysed using groups of animals after 24 h of exposure to the selected substance(s). The results indicated that agonists of the muscarinic acetylcholine and GABAA receptors and their equi-effective mixture ameliorated the negative phototactic response to fish kairomones, whereas antagonists and their mixtures increased the negative phototactism to fish kairomones. Interestingly, inhibition of the muscarinic acetylcholine receptor abolished positive phototaxis, thus inducing the phototactic response to fish kairomones. Analysis of the profile of neurotransmitters and their related metabolites showed that the D. magna behavioural responses induced by fish depend on changes in the levels of acetylcholine, dopamine and GABA.

Characterization of neurotransmitters and related metabolites in Daphnia magna juveniles deficient in serotonin and exposed to neuroactive chemicals that affect its behavior: A targeted LC-MS/MS method.

Neurotransmitters are endogenous metabolites that play a crucial role within an organism, at the chemical synapses. There is a growing interest in their analytical determination for understanding the neurotoxic effect of contaminants. Daphnia magna represents an excellent aquatic model for these environmental studies, due to its similarities with vertebrates in several neurotransmitters and related gene pathways and because of its wide application in ecotoxicological studies. Within this study, an accurate and sensible method of analysis of 17 neurotransmitters and related precursors and metabolites was developed. The method was validated in terms of sensitivity, reproducibility, precision, and accuracy, and also matrix effect was evaluated. As an independent probe of method validation and applicability, the method was applied to two different scenarios. First, it was used for the study of neurotransmitter levels in genetically mutated tryptophan hydrolase D. magna clones, confirming the absence of serotonin and its metabolite 5-HIAA. Additionally, the method was applied for determining the effects of chemical compounds known to affect different neurotransmitter systems and to alter Daphnia behavior. Significant changes were observed in 13 of the analyzed neurotransmitters across treatments, which were related to the neurotransmitter systems described as being affected by these neurochemicals. These two studies, which provide results on the ways in which the neurotransmitter systems in D. magna are affected, have corroborated the applicability of the presented method, of great importance due to the suitability of this organism for environmental neurotoxicity studies.

Changes in lipid profiles in Daphnia magna individuals exposed to low environmental levels of neuroactive pharmaceuticals.

Disruptive effects of chemicals on lipids in aquatic species are mostly limited to obesogens and vertebrates. Recent studies reported that antidepressants, anxiolytic, antiepileptic and ß-adrenergic pharmaceuticals, with putative distinct mechanisms of action at low environmental relevant concentrations, up-regulated common neurological and lipid metabolic pathways and enhanced similarly reproduction in the crustacean Daphnia magna. Conversely CRISPR mutants for the tryptophan hydrolase enzyme gene (TRH) that lack serotonin had the opposed phenotype: the lipid metabolism down-regulated and impaired reproduction. Lipid metabolism is strongly linked to reproduction in D. magna. The aim of this study is to test if the above mentioned neuro-active chemicals disrupted common lipid groups and showed also the opposed lipidomic effects as those individuals lacking serotonin. This study used ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) to study how neuro-active chemicals (carbamazepine, diazepam, fluoxetine and propranolol) at low (0.1 µg/L) and higher concentrations (1 µg/L) and three CRISPR TRH mutant clones disrupt the dynamics of glycerophospholipids and glycerolipids in Daphnia adults. Lipidomic analysis identified 267 individual lipids corresponding to three classes of glycerolipids, eleven of glycerophospholipids, one of sterols and one of sphingolipids, of which 132 and 125 changed according to the chemical treatments or across clones, respectively. Most pharmaceutical treatments enhanced reproduction whereas mutated clones lacking serotonin reproduced to a lesser extent. Except for carbamazepine, most of the tested pharmaceuticals increased some triacylglycerol species and decreased monoacylglycerols, lysophospholipids, sphingomyelins and cholesterol esters in exposed females. Opposed lipidomic pattern was observed in mutated clones lacking serotonin. Lipidomic data, thus, indicate a close link between reported transcriptomic and lipidomic changes, which are likely related to serotonin and other neurological signalling pathways.

Exposure to heavy metal-contaminated sediments disrupts gene expression, lipid profile, and life history traits in the midge Chironomus riparius.

Despite the concern about anthropogenic heavy metal accumulation, there remain few multi-level ecotoxicological studies to evaluate their effects in fluvial ecosystems. The toxicity of field-collected sediments exhibiting a gradient of heavy metal contamination (Cd, Pb, and Zn) was assessed in Chironomus riparius. For this purpose, larvae were exposed throughout their entire life cycle to these sediments, and toxic effects were measured at different levels of biological organization, from the molecular (lipidomic analysis and transcriptional profile) to the whole organism response (respiration rate, shape markers, and emergence rate). Alterations in the activity of relevant genes, as well as an increase of storage lipids and decrease in membrane fluidity, were detected in larvae exposed to the most contaminated sediments. Moreover, reduced larval and adult mass, decrease of larval respiration rate, and delayed emergence were observed, along with increased mentum and mandible size in larvae and decreased wing loading in adults. This study points out the deleterious effects of heavy metal exposure at various levels of biological organization and provides some clues regarding the mode of toxic action. This integrative approach provides new insights into the multi-level effects on aquatic insects exposed to heavy metal mixtures in field sediments, providing useful tools for ecological risk assessment in freshwater ecosystems.

Changes in lipid profiles induced by bisphenol A (BPA) in zebrafish eleutheroembryos during the yolk sac absorption stage.

Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol) has been shown to act as an obesogen and to disrupt lipid metabolism in zebrafish eleutheroembryos (ZE). To characterize the consequences of this disruption, we performed a detailed lipidomic study using ZE exposed to different BPA concentrations (0, 4, 6 and 8 mg/L of BPA) from day 2 to up to day 6 post fertilization (dpf). Total lipids at 4, 5 and 6 dpf were extracted by Folch method and analyzed by high-performance thin layer chromatography (HPTLC) as wide-range preliminary screening. Selected conditions (0 and 6 mg/L of BPA) were used to obtain a high-quality lipid profile using ultra high-performance liquid chromatography/time-of-flight mass spectrometry (UHPLC-TOFMS). BPA exposed ZE exhibited increased amounts of triglycerides (TG), diglycerides (DG), phosphatidylcholines (PC) and phosphatidylinositols (PI), regarding the control group. Analysis of time- and BPA exposure-related patterns of specific lipid species showed a clear influence of unsaturation degree (mostly in DG and PC) and/or fatty acid chain length (mostly in TG and PC derivatives) on their response to the presence of BPA. A decreased yolk-sac and energy consumption in exposed individuals appeared as the main reason for the observed BPA-driven effects. Integration of these results with previous morphological, biochemical, transcriptomic, metabolomic and behavioral data suggests a disruption of different signalling pathways by BPA that starts at very low BPA concentrations, whose effects propagate across different organization levels, and that cannot be only explained by the relatively weak estrogenic effect of BPA.

Effects of Single and Combined Low Concentrations of Neuroactive Drugs on Daphnia magna Reproduction and Transcriptomic Responses.

Assessing the risk of neuroactive pharmaceuticals in the environment requires an understanding of their joint effects at low concentrations across species. Here, we assessed reproductive and transcriptional effects of single and ternary equi-effective mixture exposure to propranolol, diazepam, and carbamazepine on the crustacean Daphnia magna at environmentally relevant concentrations. The three compounds enhanced reproduction in adults and induced specific transcriptome changes in preadolescent individuals. Comparison of the results from single exposures to a ternary equi-effective mixture of the three compounds showed additive action. Transcriptomic analyses identified 3248 genes affected by at least one of the treatments, which were grouped into four clusters. Two clusters (1897 gene transcripts in total) behaved similarly, appearing either over- or under-represented relative to control, in all single and mixture treatments. The third and fourth clusters grouped genes differently transcribed upon exposure to diazepam and propranolol, respectively. Functional transcriptomics analysis indicated that the four clusters shared major deregulated signaling pathways implicated on energy, growth, reproduction, and neurologically related processes, which may be responsible for the observed reproductive effects. Thus, our study showed additive effects at the transcriptional and physiological level and provides a novel approach to the analysis of environmentally relevant mixtures of neuroactive compounds.

Chironomus riparius exposure to field-collected contaminated sediments: From subcellular effect to whole-organism response.

The toxicity of three field-collected sediments differentially contaminated with pesticides, heavy metals, phtalates and polycyclic aromatic hydrocarbons (PAHs), was assessed in Chironomus riparius. For this purpose, C. riparius larvae were exposed throughout their entire life cycle to sediments collected in three sites along the Saulx river in France, and the toxic effects were measured at different levels of biological organization: from the molecular (lipidomic analysis and transcriptional variations) to the whole organism response (respiration rate, shape markers and emergence rate). In the sediment characterized by an intermediate level of contamination with PAHs and phtalates, we detected an increase of the cell stress response and delayed emergence of males. In the group exposed to the most contaminated sediment with PAHs, phtalates and pesticides, genes related to endocrine pathways, cell stress response and biotransformation processes were overexpressed, while female wing shape was affected. Field-collected sediment exposure did not induce significant effects on mentum shape markers or on the lipid profile. The present study provides new insights into the multilevel effects of differentially contaminated sediments in insects. This integrative approach will certainly contribute to improved assessment of the risk that complex mixtures of pollutants pose to the aquatic ecosystem.

Time-dependent transcriptomic responses of Daphnia magna exposed to metabolic disruptors that enhanced storage lipid accumulation.

The analysis of lipid disruption in invertebrates is limited by our poor knowledge of their lipidomes and of the associated metabolic pathways. For example, the mechanism by which exposure of the crustacean Daphnia magna to tributyltin, juvenoids, or bisphenol A increase the accumulation of storage lipids into lipid droplets is largely unknown/presently unclear. Here we analyze transcriptome changes subsequent to this lipid accumulation effect induced by either the pesticide pyriproxyfen (a juvenoid agonist), the plasticizer bisphenol A, or the antifouling agent tributyltin. Changes in the whole transcriptome were assessed after 8 and 24 h of exposure, the period showing the greatest variation in storage lipid accumulation. The three compounds affected similarly to a total of 1388 genes (965 overexpressed and 423 underexpressed transcripts), but only after 24 h of exposure. In addition, 225 transcripts became up-regulated in samples exposed to tributyltin for both 8 h and 24 h. Using D. melanogaster functional annotation, we determined that upregulated genes were enriched in members of KEGG modules implicated in fatty acid, glycerophospholipid, and glycerolipid metabolic pathways, as well as in genes related to membrane constituents and to chitin and cuticle metabolic pathways. Conversely, down-regulated genes appeared mainly related to visual perception and to oocyte development signaling pathways. Many tributyltin specifically upregulated genes were related to neuro-active ligand receptor interaction signaling pathways. These changes were consistent with the phetotypic effects reported in this and in previous studies that exposure of D. magna to the tested compounds increased lipid accumulation and reduced egg quantity and quality.

Allocation of glycerolipids and glycerophospholipids from adults to eggs in Daphnia magna: Perturbations by compounds that enhance lipid droplet accumulation.

Analysis of the disruptive effects of chemicals on lipids in invertebrates is limited by our poor knowledge of the lipid metabolic pathways and the complete lipidome. Recent studies shown that juvenoids and bisphenol A disrupted the dynamics of lipid droplets in the crustacean Daphnia magna. This study used ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) to study how juvenoids (pyriproxyfen and methyl farnesoate) and bisphenol A disrupt the dynamics of glycerophospholipids and glycerolipids in Daphnia adults and their allocation to eggs. Lipidomic analysis identified 234 individual lipids corresponding to three classes of glycerolipids, seven of glycerophospholipids, and one of sphingolipids, of which 194 changed according to the chemical treatments and time. Adult females in the control and bisphenol A treatment groups had low levels of triacylglycerols but high levels of glycerophospholipids, whereas those in the juvenoid treatment groups had high levels of triacylglycerols and low levels of glycerophospholipids. The opposite trend was observed for the lipid contents in the eggs produced. Because the juvenoids reduced reproduction dramatically, the females allocated less triacylglycerols to their eggs than the controls did. Interestingly, females exposed to bisphenol A allocated less triacylglycerols to their eggs despite producing a similar number of eggs as that of the controls. Thin-layer chromatography analyses confirmed the UHPLC/TOFMS results and allowed qualitative determination of cholesterol, which was also accumulated in females exposed to the juvenoids.

Fenoxycarb exposure disrupted the reproductive success of the amphipod Gammarus fossarum with limited effects on the lipid profile.

Insect growth regulator insecticides mimic the action of hormones on the growth and development of insect pests. However, they can affect the development of non-target arthropods. In the present study, we tested the effects of the growth regulator insecticide fenoxycarb on several endpoints in the freshwater crustacean Gammarus fossarum (Amphipoda). Females carrying embryos in their open brood pouch were exposed to 50 µg L-1 fenoxycarb throughout the entire oogenesis (i.e. 21 days). After exposure, newborn individuals from exposed embryos were removed from the maternal open brood pouch for lipidomic analysis, while males were added to assess the reproductive success. After fertilization, the lipid profile, energy reserve content (lipids, proteins and glycogen), and activity of phenoloxidase - an enzyme involved in the immune response - were measured in females. No significant effect of fenoxycarb exposure was observed on the lipid profile of both newborn individuals and females, while reproductive success was severely impaired in exposed females. Particularly, precopulatory behavior was significantly reduced and fertilized eggs were unviable. This study highlighted the deleterious effects of the insect growth regulator fenoxycarb on gammarid reproduction, which could have severe repercussions on population dynamics.

Analysis of the neurotoxic effects of neuropathic organophosphorus compounds in adult zebrafish.

Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN.

Study of critical points of drugs with different solubilities in hydrophilic matrices.

Hydrophilic matrices are one of the most popular controlled release systems in the world. It is well known that drug solubility increases the osmotic stress in hydrophilic matrices, resulting in higher swelling through the creation of microcavities and influencing the release rate. Drug solubility also affects the drug release mechanism, favouring the diffusion against the erosion mechanism. Nevertheless it has not been studied whether this can modify the critical points of the hydrophilic matrices. The objective of the present work is to estimate the excipient percolation threshold in HPMC K4M hydrophilic matrices containing acetaminophen, theophiline and ranitidine.HCl, and to study the influence of the drug solubility on the excipient percolation threshold. Dissolution assays were performed using the paddle method. Water uptake was examined using the modified Enslin apparatus. In order to estimate the excipient percolation threshold, the behaviour of the kinetic parameters versus the excipient volume fraction plus initial porosity, was studied. The excipient percolation thresholds were situated between 24.8-25.8, 14.7-18.4 and around 31.2% (v/v) HPMC in theophiline, ranitidine.HCl and acetaminophen matrices, respectively. On the other hand, using these and some previously reported values no relation has been found between drug solubility and excipient percolation threshold in hydrophilic matrices.

Estimation of the percolation thresholds in acyclovir hydrophilic matrix tablets.

The principles of percolation theory were applied to design controlled release matrix tablets containing acyclovir. This statistical theory studies disordered or chaotic systems where the components are randomly distributed in a lattice. The application of this theory to study the release and hydration rate of hydrophilic matrices allows to explain the changes in release and hydration kinetics of swellable matrix type controlled delivery systems. The objective of the present paper is to estimate the percolation threshold of HPMC K4M in matrices of acyclovir and to apply the obtained result to the design of hydrophilic matrices for the controlled delivery of this drug. Matrix tablets have been prepared using acyclovir as drug and HPMC K4M as matrix forming material, employing five different excipient/drug percentages. Dissolution studies were carried out using the paddle method. Water uptake measurements were performed using a modified Enslin apparatus. In order to estimate the percolation threshold, the behaviour of the kinetic parameters with respect to the excipient volumetric fraction at time zero plus initial porosity was studied. According to percolation theory, the critical points observed in dissolution and water uptake studies can be attributed to the excipient percolation threshold. This threshold was situated between between 20.76% and 26.41% v/v of excipient plus initial porosity. The knowledge of the percolation threshold of the components of the matrix formulations contributes to improve their design. First, reducing the time to market and second, increasing their robustness when they are prepared at Industrial scale, avoiding the formulation in the nearby of the percolation threshold.