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Depressing antidepressant: Fluoxetine affects serotonin neurons causing adverse reproductive responses in Daphnia magna. Bruno Campos, Claudia Rivetti, Timm Kress, Carlos Barata, and Heinrich Dircksen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00826 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 2016
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Depressing antidepressant: Fluoxetine affects
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serotonin neurons causing adverse reproductive
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responses in Daphnia magna.
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Bruno Camposa,b,†, Claudia Rivettia,b,†, Timm Kressb, Carlos Barataa, Heinrich Dircksen*b a
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Dept. of Environmental Chemistry, Institute of Environmental Assessment and Water Research
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(IDAEA-CSIC), Jordi Girona 18, E-08034, Barcelona, Spain b
Department of Zoology, Stockholm University, Svante Arrhenius väg 18A, S-106 91 Stockholm,
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Sweden
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*Corresponding Author:
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Heinrich Dircksen
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Department of Zoology, Stockholm University, Svante Arrhenius väg 18A, S-106 91 Stockholm,
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Sweden
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[email protected] 15
Phone: +46 8164076
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Fax: +46 8167715
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ABSTRACT
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Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants. As endocrine
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disruptive contaminants in the environment, SSRIs affect reproduction in aquatic organisms. In the
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water flea Daphnia magna, SSRIs increase offspring production in a food ration-dependent
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manner. At limiting food conditions, females exposed to SSRIs produce more but smaller
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offspring, which is a maladaptive life-history strategy. We asked whether increased serotonin
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levels in newly identified serotonin-neurons in the Daphnia brain mediate these effects. We
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provide strong evidence that exogenous SSRI fluoxetine selectively increases serotonin-
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immunoreactivity in identified brain neurons under limiting food conditions thereby leading to
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maladaptive offspring production. Fluoxetine increases serotonin-immunoreactivity at low food
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conditions to similar maximal levels as observed under high food conditions and concomitantly
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enhances offspring production. Sublethal amounts of the neurotoxin 5,7-dihydroxytryptamine
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known to specifically ablate serotonin-neurons markedly decrease serotonin-immunoreactivity and
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offspring production, strongly supporting the effect to be serotonin-specific by reversing the
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reproductive phenotype attained under fluoxetine. Thus, SSRIs impair serotonin-regulation of
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reproductive investment in a planktonic key organism causing inappropriately increased
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reproduction with potentially severe ecological impact.
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Keywords: Selective serotonin-reuptake inhibitor, serotonin neurons, reproduction, Daphnia
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magna, freshwater ecotoxicology
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INTRODUCTION
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Assessing the risks of long-term exposure to low doses of pharmaceuticals is an identified
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research need1, particularly for those that may act as neural and endocrine disruptors in vertebrate
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and invertebrate species2,3. Selective serotonin reuptake inhibitors (SSRIs) are currently
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antidepressants of choice widely used in psychiatric treatments of deregulated serotonin signaling
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underlying depressive disorders4. In recent years, evidence of adverse endocrine disruptive effects
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for these substances has accumulated5,6. Human populations treated with SSRIs show signs of
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developmental toxicity characterized by an increased rate of poor neonatal adaptation, reversibly
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impaired sexual function in both sexes, specifically impairing the orgasm and adverse effects on
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sperm integrity (increased DNA damage)6-8. Deregulation of serotonin signaling pathways by
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SSRIs also alters food intake and hence energy homeostasis, which may lead to obesity in
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humans9. In fish, fluoxetine, the active ingredient of Prozac®, acts as a neuroendocrine disruptor
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causing male infertility via abnormal sperm production, alters testosterone levels, feeding and
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reproductive behavior5,10.
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Daphnia magna, an important micro-crustacean in freshwater food webs3,11, is an established
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model for testing the toxicology of substances for regulatory purposes12. Daphnia shares more
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genes with humans than probably any other invertebrate, whose genome has been sequenced, and
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is an accepted model organism for human health research13. By proximate approaches, SSRIs were
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reported to change the perception of the food environment and to switch life-history responses
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towards higher food levels: females reproduced earlier, producing more but smaller offspring at
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limiting food conditions14,15. Producing more offspring of smaller size at low food conditions is
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maladaptive since smaller offspring are less fit than larger ones16. Furthermore, Campos et al.14
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have shown that the serotonin-receptor antagonist cyproheptadine reversed the effects of SSRIs at
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limiting food conditions. SSRIs also deregulated genes related to serotonin signaling pathways in 3 ACS Paragon Plus Environment
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D. magna17. This ultimately suggested that specific serotonergic neurons are involved in
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reproductive regulation and are the causative elements of SSRI action. Here, we hypothesized that
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increased levels of (synaptic) serotonin, via SSRIs action, change the perception of the food
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environment and life history responses to increased offspring production.
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In arthropods, serotonin neurons are well established18,19. They stimulate insect ecdysteroid and
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juvenile hormone production, both responsible for controlling oogenesis and vitellogenesis, and, as
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well known in decapod crustaceans, stimulate growth- and reproduction-controlling hormone
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systems20 and are involved in control of behavior and metabolism21-24. However, in non-decapod
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crustacean species, such as Daphnia, we know little about serotonin in nervous systems25 and its
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possible functions, the latter being only inferred from effects of exogenous SSRIs14,26,27. Since the
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Daphnia genome encodes serotonin biosynthesis enzymes28, serotonergic neurons involved in the
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control of growth and reproduction likely exist in D. magna.
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The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) specifically disrupts serotonergic neurons
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by oxidative damage after selective uptake. 5,7-DHT is rapidly taken up into serotonergic neurons,
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where it is oxidatively converted into the two metabolites 5-hydroxytryptamine-4,7-dione and 6,6'-
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bi(5-hydroxytryptamine-4,7-dione). The following redox reactions of these two metabolites result
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in the depletion of intraneuronal oxygen levels and OH- increase. The deleterious neurotoxicity of
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this substance is then provoked by hypoxia and OH--formation29.
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We provide first evidence that the SSRI fluoxetine (Prozac®) increases serotonin-
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immunoreactivity in newly identified optic-ganglia and brain neurons of D. magna and,
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concomitantly, increases offspring production especially under limited food conditions. In order to
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establish serotonin as a key regulator of reproduction, we manipulated this system in two
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directions (i) by increasing serotonin levels in the system via high food rations and low food ration
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plus fluoxetine and (ii) by reducing them via the specific neurotoxin 5,7-dihydroxytryptamine. The
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latter provided additional evidence for the specificity of the treatment.
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MATERIALS AND METHODS
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Chemicals. Fluoxetine hydrochloride (CAS-No 56296-78-7; analytical standard, purity 100%)
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Sigma-Aldrich, USA), 5,7-Dihydroxytryptamine creatinine sulphate (5,7-DHT; CAS No 39929-
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27-6) 1,4-Diazabicyclo[2.2.2]octane hydrochloride (Fluka, Germany), and polyclonal rabbit anti-
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serotonin serum were purchased from Sigma-Aldrich (USA, Germany). All other chemicals were
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analytical grade and obtained from Merck (Germany).
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Experimental animals. All experiments were performed using a well-characterized single clone
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of D. magna (Clone F), maintained indefinitely as pure parthenogenetic cultures12. Individual or
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bulk cultures of 10 animals/L were maintained in ASTM hard synthetic water as described
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previously16. These cultures were maintained at high food levels, fed every other day with
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Chlorella vulgaris Beijerinck (5 x 105 cell/mL), corresponding to 1.8 µg carbon/mL). C. vulgaris
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was grown axenically in Jaworski/Euglena gracilis 1:1 medium (Culture Collection of Algae and
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Protozoa, 1989; http://www.ccap.ac.uk). We changed the culture medium every other day and
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removed neonates within 24 h. Photoperiod was set to 14 h light: 10 h dark cycle and temperature
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at 20 ± 1 ºC.
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Experimental design. According to the adverse outcome pathway (AOP) framework, effects of
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pollutants on their molecular targets do not necessarily occur at the same time as they become
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apparent in individuals30. We tested if this was the case for fluoxetine and 5,7-DHT in adult
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females assayed for serotonin-immunoreactivity in the brain-optic ganglia complex and
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reproduction to assess the effect of exposure time and food ration levels.
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Under optimum conditions, the normal life cycle of D. magna can be summarized as follows: (i)
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embryonic development takes approximately 3 days at 20 ºC and >12 h of daily light, (ii) neonates
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are released as free swimming individuals, (iii) after a juvenile period of around 5 days, during
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which period animals undergo four molts, they become reproductively active adults. At this stage,
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they transfer the first eggs into the brood pouch and embryonic development starts. Thus, it takes
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approximately 8 days from birth to the first reproduction, i.e. the release of neonates. The adult
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then successively releases its broods about every 3 days followed by its ecdysis. In a
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reproductively active female, three broods are developing simultaneously: one clutch of eggs
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develops in the brood pouch, while the second is provisioned in the ovaries, and the third clutch
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differentiates from nurse cells to oocytes31. This means that effects on egg provisioning (when the
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SSRI effect is elicited) and, hence, on the total reproductive output require at least 6 days (two
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consecutive broods) to be reliably measurable as offspring production. SSRIs increase the rates of
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juvenile development and offspring production in D. magna in a food-dependent manner14,15. For
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the AOP framework, the timing of events occurring at the molecular level on primary targets and
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those measured at the individual level (i.e. reproduction) is crucial to link the initiating with the
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key events30. In this regard, we designed four assays to study exposure to 40 µg/L of fluoxetine
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under different ontogenetic periods (see design summary in Figure 1). We previously reported that
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effects of fluoxetine and of other SSRIs enhancing offspring production also occurred at lower
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concentrations (3-10 µg/L)14,15, which were closer to those reported in the field32,33. Here we used
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the concentration affecting mostly the reproduction, facilitating the establishment of links between
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molecular and reproductive responses.
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In assay 1, we exposed juveniles from birth until the first release of neonates (~8 days). In assays
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2, 3, and 4, reproductive females were exposed during one, two and three consecutive broods, i.e.,
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approximately 3, 6 and 9 days, respectively (Figure 1). The first batch of neonates (hatching within 6 ACS Paragon Plus Environment
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the first 48 - 72 h) was always discarded and not evaluated as these animals were not exposed to
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the tested chemicals during their egg provisioning period12. Thus, we counted only neonates from
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the second and third broods to assess effects on total offspring production. This means that in
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females exposed during 6 and 9 days (in assays 3 and 4, respectively, but not in assay 2) we
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counted the number of neonates released in one and two consecutive clutches, respectively. These
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assays were performed at low (1*105 cells/mL) and high food levels (5*105 cells/mL) using
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C. vulgaris as the sole food source14. The high food level equals ad libitum or surplus food supply,
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while the low food level is limiting though sufficient to allow the animals to develop and
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reproduce. The purpose of assays 2, 3, and 4 was to assess the temporal dynamics in the primary
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target of SSRIs, serotonin immunoreactivity in the brain, across different food conditions (with or
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without SSRIs). Specifically, when adult animals reared at high food levels were switched to low
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food with and without fluoxetine, this treatment was key to the understanding of when serotonin
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depletion occurred, if any, and was affected by fluoxetine exposure. Assays 1, 3, 4 allowed to
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relate serotonin immunoreactivity with the reproductive effects. A negative control via specific
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chemical ablation of serotonin-neurons by 5,7-DHT was used in order to provide the two-
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directional manipulations (see above), which lent strong support towards the specificity of the
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effects. We used 1 and 10 µg/L 5,7-DHT to affect serotonin-immunoreactivity and reproduction in
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adult females exposed for a short time (6 h) or from birth to first reproduction. These tests were
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performed at high food ration level only.
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Exposure assays were initiated with neonates (
