Antidepressants - The new endocrine disruptors? The case of crustaceans.

Antidepressants are high-volume pharmaceuticals that accumulate to concentrations in the µg·L-1 range in surface waters. The release of peptide hormones via neurosecretory cells appears as a natural target for antidepressants. Here I review research that suggests that antidepressants indeed disrupt endocrine signalling in crustaceans, by acting on the synthesis and release of neurohormones, such as crustacean hyperglycaemic hormone, moult inhibiting hormone and pigment dispersing hormone in decapods, as well as methyl farnesoate in Daphnids. Hence, antidepressants can affect hormonal regulation of physiological functions: increase in energy metabolism and activity, lowered ecdysteroid levels, potentially disrupting moult and somatic growth, reducing colour change capacity and compromising camouflage, as well as induction of male sex determination. Several studies further suggest effects of antidepressants on crustacean reproduction, but the hormonal regulation of these effects remains elusive. All things considered, a body of evidence strongly suggests that antidepressants are endocrine disrupting compounds in crustaceans.

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass.

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.

Cuttlefish color change as an emerging proxy for ecotoxicology.

Lately, behavioral ecotoxicology has flourished because of increasing standardization of analyses of endpoints like movement. However, research tends to focus on a few model species, which limits possibilities of extrapolating and predicting toxicological effects and adverse outcomes at the population and ecosystem level. In this regard, it is recommended to assess critical species-specific behavioral responses in taxa playing key roles in trophic food webs, such as cephalopods. These latter, known as masters of camouflage, display rapid physiological color changes to conceal themselves and adapt to their surrounding environments. The efficiency of this process depends on visual abilities and acuity, information processing, and control of chromatophores dynamics through nervous and hormonal regulation with which many contaminants can interfere. Therefore, the quantitative measurement of color change in cephalopod species could be developed as a powerful endpoint for toxicological risk assessment. Based on a wide body of research having assessed the effect of various environmental stressors (pharmaceutical residues, metals, carbon dioxide, anti-fouling agents) on the camouflage abilities of juvenile common cuttlefish, we discuss the relevance of this species as a toxicological model and address the challenge of color change quantification and standardization through a comparative review of the available measurement techniques.

Proteomic changes in the extracellular environment of sea bass thymocytes exposed to 17α-ethinylestradiol in vitro.

The thymus is an important immune organ providing the necessary microenvironment for the development of a diverse, self-tolerant T cell repertoire, which is selected to allow for the recognition of foreign antigens while avoiding self-reactivity. Thymus function and activity are known to be regulated by sex steroid hormones, such as oestrogen, leading to sexual dimorphisms in immunocompetence between males and females. The oestrogenic modulation of the thymic function provides a potential target for environmental oestrogens, such as 17α-ethynylestradiol (EE2), to interfere with the cross-talk between the endocrine and the immune system. Oestrogen receptors have been identified on thymocytes and the thymic microenvironment, but it is unclear how oestrogens regulate thymic epithelial and T cell communication including paracrine signalling. Much less is known regarding intrathymic signalling in fish. Secretomics allows for the analysis of complex mixtures of immunomodulatory signalling factors secreted by T cells. Thus, in the present study, isolated thymocytes of the European sea bass, Dicentrarchus labrax, were exposed in vitro to 30 nM EE2 for 4 h and the T cell-secretome (i.e., extracellular proteome) was analysed by quantitative label-free mass-spectrometry. Progenesis revealed a total of 111 proteins differentially displayed between EE2-treated and control thymocytes at an α-level of 5% and a 1.3-fold change cut off (n = 5-6). The EE2-treatment significantly decreased the level of 90 proteins. Gene ontology revealed the proteasome to be the most impacted pathway. In contrast, the abundance of 21 proteins was significantly increased, with cathepsins showing the highest level of induction. However, no particular molecular pathway was significantly altered for these upregulated proteins. To the best of our knowledge, this work represents the first study of the secretome of the fish thymus exposed to the environmental oestrogen EE2, highlighting the impact on putative signalling pathways linked to immune surveillance, which may be of crucial importance for fish health and defence against pathogens.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

Antidepressants Modify Cryptic Behavior in Juvenile Cuttlefish at Environmentally Realistic Concentrations.

Contamination of the marine environment by antidepressants may affect neurophysiological processes in nontarget organisms, such as the common cuttlefish, Sepia officinalis. The present study tested whether environmentally realistic concentrations of antidepressants, that is, fluoxetine alone (5 ng L-1 ) or cumulated with venlafaxine (2.5 or 5 ng L-1 ), affect camouflage in newly hatched cuttlefish. The results show that antidepressants improved uniform body patterns, whereas disruptive body patterns were not affected. Environ Toxicol Chem 2021;40:2571-2577. © 2021 SETAC.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

Alteration of predatory behaviour and growth in juvenile cuttlefish by fluoxetine and venlafaxine.

Antidepressants in coastal waters may affect ontogeny of predatory behaviour in cuttlefish, which may, as a result, affect growth of newly-hatched cuttlefish. We investigated the effects of two of the most prescribed antidepressants, fluoxetine (FLX) and venlafaxine (VEN) in environmentally realistic concentrations on the predatory behaviour of hatchlings of Sepia officinalis. Newly-hatched cuttlefish were exposed from 1 h (i.e., day 1) to 5 days after hatching to either FLX alone (5 ng·L-1) or combined with VEN (2.5 ng·L-1 or 5 ng·L-1 each) to simulate an environmentally realistic exposure scenario. Their predatory behaviour was analysed through several parameters: prey detection, feeding motivation and success in catching the prey. All parameters improved in control animals over the first five days. The combination of FLX and VEN at 5 ng·L-1 each altered the predatory behaviour of the hatchlings by increasing the latency before attacking the prey, i.e., reducing feeding motivation, as well as by reducing the number of successful attacks. The changes in predatory behaviour tended to reduce food intake and affected growth significantly at 28 days post-hatching. Exposures to either FLX at 5 ng·L-1 or FLX and VEN in mixture at 2.5 ng·L-1 each tended to produce similar effects, even though they were not statistically significant. It is likely that the antidepressants affect maturation of the predatory behaviour and/or learning processes associated with the development of this behaviour. The slightest delay in maturation processes may have detrimental consequences for growth and population fitness.

A Crab Is Not a Fish: Unique Aspects of the Crustacean Endocrine System and Considerations for Endocrine Toxicology.

Crustaceans-and arthropods in general-exhibit many unique aspects to their physiology. These include the requirement to moult (ecdysis) in order to grow and reproduce, the ability to change color, and multiple strategies for sexual differentiation. Accordingly, the endocrine regulation of these processes involves hormones, receptors, and enzymes that differ from those utilized by vertebrates and other non-arthropod invertebrates. As a result, environmental chemicals known to disrupt endocrine processes in vertebrates are often not endocrine disruptors in crustaceans; while, chemicals that disrupt endocrine processes in crustaceans are often not endocrine disruptors in vertebrates. In this review, we present an overview of the evolution of the endocrine system of crustaceans, highlight endocrine endpoints known to be a target of disruption by chemicals, and identify other components of endocrine signaling that may prove to be targets of disruption. This review highlights that crustaceans need to be evaluated for endocrine disruption with consideration of their unique endocrine system and not with consideration of the endocrine system of vertebrates.

Effects of environmental antidepressants on colour change and locomotor behaviour in juvenile shore crabs, Carcinus maenas.

Juvenile crabs of Carcinus maenas thrive in coastal waters reputed to be the receptacle of continental pollution. Amongst the many pollutants encountered, antidepressants, such as fluoxetine (FLX) and venlafaxine (VEN), often detected at the ng•L-1 range, are particularly worrying because of their action on the levels of monoamines, such as serotonin, noradrenaline and dopamine. In crustaceans, those monoamines are involved in colour change through their action on neuropeptide hormones. In addition, they are known to have a role in different behaviours, such as locomotion. Both colour change and locomotion are strategies used by juvenile crabs to hide and escape from predators. To investigate if the presence of antidepressants may alter behaviours of ecological importance, juvenile crabs were exposed to environmentally realistic concentrations of either 5 ng•L-1 of FLX alone or in combination with VEN at 5 ng•L-1. The ability to change colour depending on the environment and the locomotor activity of juvenile crabs were monitored weekly over 25 days. Animals exposed to antidepressants displayed a different pattern of colour change than the controls, especially those exposed to the combination of FLX and VEN at 5 ng•L-1 each, and were less efficient to adapt to their environment, i.e., they were not as pale and not as dark as controls or crabs exposed to FLX at 5 ng•L-1. Moreover, juvenile crabs exposed to the combination of antidepressants exhibited an enhanced locomotor activity throughout the exposure period with a higher velocity and distance moved as well as more time spend moving. The alteration of cryptic behaviours, such as colour change and locomotion by antidepressants persistently present in marine environment at low concentrations may have an impact on the survival of juvenile of C. maenas on the long term.

The influence of 17ß-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax.

The female sex steroid 17ß-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Nevertheless, little is known about the function of oestrogen in immune system development. To further the understanding of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2·L-1, at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng·L-1 was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αß T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is oestrogen-sensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.

Hidden in the sand: Alteration of burying behaviour in shore crabs and cuttlefish by antidepressant exposure.

Pharmaceuticals such as antidepressants are constantly released into the aquatic environment. Consequently, fluoxetine (FLX) and venlafaxine (VEN), the active molecules of Prozac© and Effexor©, are detected up to several µg.L-1 in freshwater and marine coastal waters. Both compounds act on the serotoninergic system, which may result in behavioural impairment, especially in juvenile animals presumed to be more susceptible to low concentrations than adults. The objective of this study was to determine whether environmental concentrations of FLX alone or combined with VEN modulate innate burying behaviour in two juvenile marine invertebrates, i.e. Sepia officinalis and Carcinus maenas. Juvenile cuttlefish were exposed from hatching to 30 days post-hatching to either FLX alone (i.e. 5 ng.L-1) or in mixture with VEN (i.e. either 2.5 ng.L-1 or 5 ng.L-1 of each antidepressant). Juvenile crabs (<2 cm carapace width) were exposed for a period of 22 days to 5 ng.L-1 of FLX and a mixture of 5 ng.L-1 of FLX and VEN each. Several parameters of sand-digging behaviour were analysed weekly in both species. The occurrence of sand-digging behaviour decreased in cuttlefish exposed to a mixture of FLX and VEN at the lowest concentration (2.5 ng.L-1 each). Because sand-digging behaviour improved in controls, this decrease was likely to be related to a modification of maturation and/or learning processes. At the mixture of 5 ng.L-1 VEN and FLX each, a better body covering was observed in juvenile crabs. In both species, innate behaviour was modified under exposure to mixtures of FLX and VEN at environmentally realistic concentrations. These alterations were observed at an early developmental stage, when animals are particularly prone to predation. Hence, modified maturation of behavioural traits and, putatively, learning processes by exposure to pseudo-persistent antidepressants may affect the survival of these two species in the long term.

Oestrogen differentially modulates lymphoid and myeloid cells of the European sea bass in vitro by specifically regulating their redox biology.

Besides their obvious role in sex determination and reproduction, oestrogens display a prominent and complex immunomodulatory role across all vertebrates. To date, our knowledge on the oestrogenic immunomodulation in non-mammalian species is, however, scarce. In both teleosts and mammals, the direct immunomodulatory function of oestrogen is underscored by the presence of multiple oestrogen receptor subtypes in the various immune cells. For a better understanding of the regulatory processes, we investigated the oestrogen receptor expression in two major lymphoid organs of European sea bass: the head-kidney and the spleen. All oestrogen receptor subtypes, including nuclear and membrane oestrogen receptors, were present in both immune organs as well as in the isolated leucocytes. The same findings have been previously made for the thymus. To determine the oestrogen responsiveness of the different immune cell populations and to evaluate the importance of non-genomic and genomic pathways, we assessed the kinetics and the concentration dependent effects of 17ß-oestradiol on isolated leucocytes from the head-kidney, the spleen and the thymus in vitro. Given the importance of reactive oxygen species as signalling and defence components in mammalian immune cells, the oxidative burst capacity, the redox status and the viability of both lymphoid and myeloid cells were measured by flow cytometry. The treatment with 17ß-oestradiol specifically modulated these parameters depending on (1) the time kinetic, (2) the concentration of 17ß-oestradiol, (3) the immune cell population (lymphoid and myeloid cells) as well as (4) the lymphoid organs from which they originated. The observed in vitro oestrogenic effects as well the presence of various oestrogen receptor subtypes in the immune cells of sea bass suggest a complex and direct oestrogenic action via multiple interconnected oestrogen-signalling pathways. Additionally, our study suggests that the oestrogenic regulation of the sea bass immune function involves a direct and tissue specific modulation of the immune cell redox biology comprising redox signalling, NADPH-oxidase activity and H2O2-permeability, thus changing oxidative burst capacity and immature T cell fate because oestrogen impacted thymocyte viability. Importantly, immune cells from both primary and secondary lymphoid organs have shown specific in vitro oestrogen-responsiveness. As established in mammals, oestrogen is likely to be specifically and directly involved in immature T cell differentiation and mature immunocompetent cell function in sea bass too.

In vivo effects of serotonin and fluoxetine on cardio-ventilatory functions in the shore crab Carcinus maenas (L. 1758).

Serotonin (5-HT) takes a key position in regulating vital functions, such as cardio-ventilatory activity, locomotion and behaviour. Selective serotonin reuptake inhibitors (SSRIs) modulate the serotonergic system and thus affect these functions. Rhythmic behaviours, such as cardio-ventilatory activity, are controlled by central pattern generators, which in turn are regulated by 5-HT. In crustaceans, 5-HT also regulates the synthesis and secretion of crustacean hyperglycaemic hormone, a pleiotropic hormone involved in the mobilisation and release of glucose into the haemolymph, thus stimulating the animal's activity. As a matter of consequence, SSRIs may affect cardio-ventilatory activity. In order to examine how the SSRIs affect fundamental physiological parameters based on rhythmic behaviours in decapods, cardio-respiratory activity in the shore crab Carcinus maenas was assessed after pericardial injection of a single dose of either 0.5 µM, 0.75 µM or 1 µM fluoxetine, respectively. Simultaneous recordings of heart and scaphognathite movements in both brachial chambers were conducted by measuring impedance changes in the respective body compartments. Injection of 5-HT had an immediate effect on cardio-ventilatory activities and strongly upregulated both cardiac and ventilatory activities. Fluoxetine showed similar effects, entailing moderate tachycardia and increased ventilation rates. Compared to 5-HT, these effects were delayed in time and much less pronounced. Metabolism of fluoxetine into the active compound nor-fluoxetine might account for the delayed action, whereas compensatory regulation of cardio-ventilatory frequencies and amplitudes are likely to explain the attenuation of the responses compared to the strong and immediate increase by 5-HT. Overall, the results suggest increased 5-HT levels in invertebrates following fluoxetine exposure, which are able to disturb physiological functions regulated by 5-HT, such as cardiac and respiratory activity.

Oestrogen, an evolutionary conserved regulator of T cell differentiation and immune tolerance in jawed vertebrates?

In teleosts, as in mammals, the immune system is tightly regulated by sexual steroid hormones, such as oestrogens. We investigated the effects of 17ß-oestradiol on the expression of several genes related to T cell development and resulting T cell subpopulations in sea bass, Dicentrarchus labrax, for a primary lymphoid organ, the thymus, and two secondary lymphoid organs, the head-kidney and the spleen. In parallel, the oxidative burst capacity was assessed in leucocytes of the secondary lymphoid organs. Apoptosis- and proliferation-related genes, indicative of B and T cell clonal selection and lymphoid progenitor activity, were not affected by elevated oestrogen-levels. Sex-related oestrogen-responsiveness in T cell and antigen-presenting cell markers was observed, the expression of which was differentially induced by oestrogen-exposure in the three lymphoid organs. Remarkably, in the spleen, oestrogen increased regulatory T cell-related gene expression was associated with a decrease in oxidative burst capacity. To the best of our knowledge, this study indicates for the first time that physiological levels of oestrogen are likely to promote immune tolerance by modulating thymic function (i.e., T cell development and output) and peripheral T cells in teleosts, similar to previously reported oestrogenic effects in mammals.

Relevance of body size and shell colouration for thermal absorption and heat loss in white garden snails, Theba pisana (Helicidae), from Northern France.

The internal temperature of land snails depends on environmental factors, such as exposure to electromagnetic radiation and airflow as well as biotic factors including shell size, shell colouration and thickness or the resting position of the snail. In controlled field experiments, we quantified heating by thermal absorption of light and airflow-induced heat loss in the white garden snail, Theba pisana, from Normandy, France. Heating experiments revealed a significant positive relation of the internal body temperature with illumination period, shell temperature and air temperature at different times of day. The size of the snails was negatively related with both of the given illumination times: smaller animals heated up stronger than larger ones. The temperature at the surface of the shell significantly depended on the illumination period and the time of day. An AIC-based quality assessment of multiple linear modelling showed that, for explaining both shell surface and internal temperature of the soft body, several factors, i.e., exposure time, daytime, shell size and colouration contributed to the best models, respectively. Similarly, heat loss of the soft body after and during exposure of the snails to sunlight by a constant airflow depended on the initial body temperature, shell size, colouration and ambient air temperature. Our study revealed also the importance of both shell size and colouration for the loss of body temperature under natural conditions: small and banded animals that had heated up to temperatures above 30°C cooled down faster than large and un-banded ones.

Toxicokinetics, disposition and metabolism of fluoxetine in crabs.

The disposition and metabolism of fluoxetine in the European shore crab and the Dungeness crab were assessed. Crabs received intracardiac doses of either 0.13 µg/kg or 0.5 mg/kg fluoxetine, respectively. In addition, fluoxetine was administered to Metacarcinus cancer by oral gavage at 7.8 mg/kg. The distribution of fluoxetine was quantified in haemolymph and digestive gland for both crabs, as well as brain, muscle, and testis of Carcinus maenas, over 12 days. The metabolite norfluoxetine, was also measured in C. maenas. Fluoxetine was mainly found in lipid rich tissues. Distribution coefficients increased for digestive gland until three days after fluoxetine administration and then decreased until the end of the observations. The highest distribution coefficients were obtained for brain. Norfluoxetine displayed continuously high levels in digestive gland and brain. The strong decrease in fluoxetine and the concomitant increase in norfluoxetine demonstrates that decapod crustaceans metabolise fluoxetine into the more biologically active norfluoxetine. Fluoxetine levels in the haemolymph of M. cancer declined within 20 h, but showed a second peak 25 h later, suggesting remobilisation from tissues sequestering the compound. The steady state volume distribution and the total body clearance of fluoxetine were high, consistent with high diffusion of fluoxetine into the peripheral tissues and biotransformation as an important elimination pathway. Oral administration of fluoxetine prolonged its half-life in M. cancer, but bioavailability was low. These results confirm the high distribution into nervous tissue, extensive biotransformation into the highly active norfluoxetine and a half-life similar to that observed in vertebrates.

Oestrogen receptor distribution related to functional thymus anatomy of the European sea bass, Dicentrarchus labrax.

In jawed vertebrates, the crosstalk between immune and endocrine system as well as many fundamental mechanisms of T cell development are evolutionary conserved. Oestrogens affect mammalian thymic function and plasticity, but the mechanisms of action and the oestrogen receptors involved remain unclear. To corroborate the oestrogenic regulation of thymic function in teleosts and to identify the implicated oestrogen receptor subtypes, we examined the distribution of nuclear and membrane oestrogen receptors within the thymus of the European Sea bass, Dicentrarchus labrax, in relation to its morpho-functional organisation. Immunohistological analysis specified thymus histology and organisation in teleosts and described, for the first time, Hassall's corpuscle like structures in the medulla of sea bass. All oestrogen receptors were expressed at the transcript and protein level, both in T cells and in stromal cells belonging to specific functional areas. These observations suggest complex regulatory actions of oestrogen on thymic function, notably through the stromal microenvironment, comprising both, genomic and non-genomic pathways that are likely to affect T cell maturation and trafficking processes. Comparison with birds, rodents and humans supports the thymic localization of oestrogen receptors and suggests that oestrogens modulate T cell maturation in all gnathostomes.

Neuroendocrine disruption in the shore crab Carcinus maenas: Effects of serotonin and fluoxetine on chh- and mih-gene expression, glycaemia and ecdysteroid levels.

Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2h and 12h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.

Sensitive periods for 17ß-estradiol exposure during immune system development in sea bass head kidney.

An increasing body of evidence suggests that sex steroids play an important role in the development and regulation of vertebrate immune defense. Therefore, compounds with estrogenic activity may influence the immune system via receptor-mediated pathways. The presence of estrogen receptors in immune cells and organs during the early stages of development may indicate that female steroid hormones are involved in the maturation of the fish immune system. This is of particular importance, as some marine fish are probably exposed to sources of exogenous estrogens while they reside in their estuarine nursery grounds. In this study, the influence of 17ß-estradiol (E2) on estrogen receptor and cytokine gene expression was assessed in juvenile sea bass (Dicentrarchus labrax) together with characterization of the head kidney leukocyte populations and corresponding phagocytic activity during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1ß and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation in the sea bass head kidney. The E2 treatment of 120 dph fish led to an increase in estrogen receptor ß2 and a decrease in transforming growth factor ß1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages. Additionally, these changes were observed during developmental periods described as critical phases for B-lymphocyte development in mammals. Consequently, exogenous estrogens have the potential to modify the innate immune response in juvenile sea bass and to exert detrimental effects on head kidney development. Copyright © 2015 John Wiley & Sons, Ltd.