Identification of metabolic pathways in Daphnia magna exposed to selective serotonin reuptake inhibitors using transcriptomic, immunocytochemistry and physiological responses.
ABSTRACT: Assessing the risks of long-term exposure to low doses of pharmaceuticals is an identified research need, particularly for those that may act as neural disruptors in non-vertebrate species. Selective serotonin reuptake inhibitors (SSRIs) act by blocking the re-uptake of serotonin in the nerve synapses, increasing the effective concentration of serotonin in the intra-synaptic space and therefore stimulating serotoninergic neurons. This effect is used worldwide to treat clinical depression in humans, with the consequence of their widespread release into the environment. SSRIs have been found to alter the reproductive physiology of D. magna and other invertebrates in a biphasic way. Low levels of fluoxetine stimulated offspring production in Daphnia magna and Ceriodaphnia dubia at 36 and 50 µg/l, respectively, but higher exposure levels inhibited reproduction in the same species. In this study we explore the hypothesis that SSRI can affect D. magna juvenile developmental rates and offspring production disrupting serotonin activity that is known to regulate carbohydrate and oxidative metabolic pathways. The primary mode of action was tested using transcriptomic analyses of juveniles and adults exposed to SSRIs (fluoxetine 80 ppbs, fluovoxamine 30 ppbs) and serotonin-immunocytochemistry assays of D. magna brains. Reproduction, respirometry and biochemical measurements allowed to related gene and immunological base effects to phenotypic responses.
Project description:Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 technology was used to generate seven indel TRH mutants in Daphnia magna. Mono-allelic indel TRH-/+ clones showed normal levels of serotonin, measured by both immunohistochemistry and mass spectrometry (LC-MS/MS), whereas bi-allelic indel TRH-/- clones showed no detectable levels of serotonin. Life history and behavioral responses of TRH-/- clones showed the anti-phenotype of those exposed to selective serotonin reuptake inhibitors (SSRI). Mutants lacking serotonin grew less and hence reproduced latter, produced smaller clutches of smaller offspring and responded to a greater extent to light than wild type individuals. Mono-allelic indel TRH-/+ individuals showed the intermediate phenotype. The SSRI fluoxetine enhanced offspring production in all clones and decreased the response to light only in those clones having serotonin, thus indication that behavioral effects of this drug in D. magna are associated to serotonin. Results obtained with the TRH mutants are in line with reported ones in TRH knockouts of Caenorhabditis elegans, Drosophila and mice, indicating that there is one gene encoding TRH, which is the serotonin limiting enzyme in both the central and the periphery nervous system in Daphnia and that deprivation of serotonin increases anxiety-like behavior.
Project description:Microarray Construction The D. magna microarray was constructed using DNA fragments from three sources: i) Stress-specific cDNAs were generated using Selective Subtractive Hybridisation (SSH) on organisms exposed to the stressors shown in Table 1. SSH was performed with forward and reverse subtraction using a PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc, USA). cDNA was prepared from mRNA extracted from batches of 100 D. magna, 48 h old, using a Straight Aâ¬s mRNA Isolation Kit System (Novagen Inc., UK) (2,029 clones). ii) ESTs obtained from a cDNA library from unexposed mixed aged organisms obtained from a 3-4 week old culture (Watanabe et al. 2005) (10,272 clones). iii) cDNAs isolated following an SSH, as above, between 15 adults carrying eggs and 75 juveniles (Soetaert et al. 2006) (1,143 clones). Gene fragment annotation Sequencing of the 3,172 SSH ESTs was carried out with M13-Long primers. Blast searches were performed on specific fragments that responded significantly to the exposure treatment. Sequences were annotated according to BlastX homology search against Uniprot Swissprot (http://www.ebi.ac.uk/swissprot) and Uniprot Trembl (invertebrate section, http://www.ebi.ac.uk/trembl) March 2006. Sequences were only annotated if they were found to have a Blast hit with the expect value smaller than 0.00001 and a score above 50. GeneBank/UniProt Accession number and speciesâ¬ match were recorded with each annotation. We printed PCR products generated from the above, onto glass slides. All spots have been sequenced except for (iii), only a few available and some sequencing failed for various reasons. Therefore we have the sequences for around 90% of the spots. BLAST searches revealed a large proportion (nr 60%) had no significant match with existing entries in GenBank/Uniprot/Trembl. Because of low confidence we've not considered them, otherwise this would lead to huge and erroneous assumptions. All (i and ii) sequences are available either on GenBank or on our website. All the spots that came out as significant on our experiments were reviewed and only those that have been annotated, with significant BLAST match, are being published, with respective Accession numbers, species match and E-values. Fragments were therefore described as â¬Sunnanotatedâ¬ý where no significant difference was measured or â¬Sno significant matchâ¬ý for those that were significant but no matches were found. Significant genes were annotated respectively as described above. Dose Reponse Tests were carried out using the developed microarray on cadmium, ph, lufenuron, calcium limitation and ibuprofen; details below: Cadmium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of cadmium chloride from 0, 10, 33 to 60 µg L-1 (corresponding to 0, 6.13, 20.24 and 36.79 µg L-1 Cd2+), in quadruplicates. pH: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to pH ranging from 8.0 (controls), 5.5, 5.3 and 5.2, in quadruplicates. pH was maintained by adding HCl on a daily basis. Lufenuron: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of C14 labelled, lufenuron ranging from 0, 0.35, 0.59 to 1.00 ug L-1, in quadruplicates. Calcium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to calcium chloride limitation ranging from 195.87 (culture condition), 20, 10 and 5 mg L-1 (CaCo3), in quadruplicates. Ibuprofen: RNA was obtained from populations of 300 D. magna, 24-48 h old, exposed for 24 h to ibuprofen (IB) sodium; 0 (control), 20, 40 and 80 mg L-1, in *quadruplicates. *One replicate at 80 mg L-1 was lost, thus this treatment was done in triplicate. IB was measured as the pharmacological active ingredient. Juvenile D. magna were used in order to restrict the response to genes associated with stress responses and growth, rather than genes associated with different stages of reproduction. Following exposure, daphnids were stored in RNAlater (Ambion, UK) at -80°C.
Project description:Juvenile (<24hr old) Daphnia magna populations (n=25) were exposed (24hr) to 0.00ug, 0.15ug, 0.33ug, 0.65ug lufenuron Benzoylphenylurea growth inhibitor. Transcriptomic responses were screened using a D.magna cDNA microarray and the regulation of gene expression via log fold-change in transcript abundance, assessed. Genes involved in the synthesis of chitin were expected to respond to lufenuron, given the mode of action. Extracted total RNA was amplified (400ng/sample) prior to hybridisation with the expectation that this would increase the abundance of significantly responding genes.
Project description:Juvenile (<24hr old) Daphnia magna populations (n=250) were exposed (24hr) to Benzoylphenylurea growth inhibitor (14C labeled) lufenuron. Treatments were 0.15 ug/L, 0.33 ug/L, 0.65 ug/L lufenuron solvent control was 100 ul/L acetone carrier . Transcriptomic responses were screened using a D.magna cDNA microarray and the regulation of gene expression via log fold-change in transcript abundance, assessed. Genes involved in the synthesis of chitin were expected to respond to lufenuron, given the mode of action
Project description:The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.
Project description:We isolated two mutants defective in the uptake of exogenous serotonin (5-HT) into the neurosecretory motor neurons of Caenorhabditis elegans. These mutants were hypersensitive to exogenous 5-HT and hyper-responsive in the experience-dependent enhanced slowing response to food modulated by 5-HT. The two allelic mutations defined the gene mod-5 (modulation of locomotion defective), which encodes the only serotonin reuptake transporter (SERT) in C. elegans. The selective serotonin reuptake inhibitor fluoxetine (Prozac) potentiated the enhanced slowing response, and this potentiation required mod-5 function, establishing a 5-HT- and SERT-dependent behavioral effect of fluoxetine in C. elegans. By contrast, other responses of C. elegans to fluoxetine were independent of MOD-5 SERT and 5-HT. Further analysis of the MOD-5-independent behavioral effects of fluoxetine could lead to the identification of novel targets of fluoxetine and could facilitate the development of more specific human pharmaceuticals.
Project description:Fluoxetine is a selective serotonin reuptake inhibitor that increases serotonin concentration in the central nervous system and modulates various systems, including the control of sympathetic outflow and the hypothalamus-pituitary-adrenal. However, it is not yet established whether fluoxetine can modulate the responses to stressors stimulants (physical or chemical) that trigger cortisol response in zebrafish. We demonstrate that fluoxetine blunts the response to physical stress, but not to chemical stress.
Project description:There is growing use of psychostimulant cognitive enhancers such as methylphenidate (Ritalin). Methylphenidate differs from the psychostimulant cocaine because it does not enhance synaptic levels of serotonin. We investigated whether exposure to methylphenidate combined with a serotonin-enhancing medication, the prototypical selective serotonin reuptake inhibitor (SSRI) fluoxetine (Prozac), would produce more "cocaine-like" molecular and behavioral changes.We measured the effects of fluoxetine on gene expression induced by the cognitive enhancer methylphenidate in the striatum and nucleus accumbens of rats, by in situ hybridization histochemistry. We also determined whether fluoxetine modified behavioral effects of methylphenidate.Fluoxetine robustly potentiated methylphenidate-induced expression of the transcription factors c-fos and zif 268 throughout the striatum and to some degree in the nucleus accumbens. Fluoxetine also enhanced methylphenidate-induced stereotypical behavior.Both potentiated gene regulation in the striatum and the behavioral effects indicate that combining the SSRI fluoxetine with the cognitive enhancer methylphenidate mimics cocaine effects, consistent with an increased risk for substance use disorder.
Project description:BACKGROUND:The potential long-term effects of childhood fluoxetine therapy on brain serotonin systems were studied using a nonhuman primate model, the rhesus monkey. METHODS:Juvenile male rhesus (1-4 years of age, corresponding to 4-11 years of age in children) were treated orally with fluoxetine (2 mg/kg) or vehicle daily for 2 years and removed from treatment during the third year. Each treatment group was assigned an equal number of subjects with low and high transcription polymorphisms of MAOA. One year after discontinuation of treatment, positron emission tomography scans were conducted (n = 8 treated monkeys, n = 8 control monkeys) using [11C]DASB to quantify serotonin transporter in 16 cortical and subcortical regions. RESULTS:Fluoxetine-treated monkeys with MAOA low transcription polymorphism had significantly lower [11C]DASB binding potentials than control monkeys. This finding was seen throughout the brain but was strongest in prefrontal and cingulate cortices. The MAOA × fluoxetine interaction was enhanced by binding potentials that were nonsignificantly higher in monkeys with high transcription polymorphism. CONCLUSIONS:Juvenile fluoxetine treatment has residual posttreatment effects on brain serotonin transporter that depend on MAOA genotype. MAOA genotype may be important to consider when treating children with fluoxetine.
Project description:Lactation causes dramatic maternal calcemic adaptations where up to 10% of maternal bone density is mobilized for milk calcium. Serotonin plays a role in regulating this calcemic demand through alterations in DNA methylation. Selective serotonin reuptake inhibitors used to treat peripartum depression exacerbate this signaling. The first objective of this study was to determine the impact of fluoxetine, one of the most popular selective serotonin reuptake inhibitors, on the transcriptome of the mouse mammary gland during peak lactation. The second objective was to determine whether supplementation with folic acid, a well-known methyl donor, would mitigate the gene expression changes induced by fluoxetine. Overall design: Two weeks before mating, C57BL/6 female mice were randomly assigned into breeder diet (4mg/kg folic acid, n = 16) or folic acid supplemented diet (24 mg/kg folic acid, n = 16). Female mice were bred overnight, and pregnancy was determined by observing a seminal plug, at which time the mice were housed individually and randomly assigned to fluoxetine or saline treatment. This resulted in 4 treatments (n = 8 mice per treatment): breeder diet + saline, breeder diet + fluoxetine, folic acid supplemented diet + saline, or folic acid supplemented diet + fluoxetine. Beginning on embryonic day 13 of pregnancy through day 10 of lactation, dams received a daily intraperitoneal injection of either 20 mg/kg bodyweight fluoxetine hydrochloride or saline. At day 10 of lactation, dams were euthanized, and mammary glands were rapidly extracted and stored at -80°C until RNA extraction.