Developmental fluoxetine exposure in zebrafish reduces offspring basal cortisol concentration via life stage-dependent maternal transmission

Abstract

Fluoxetine (FLX) is a pharmaceutical used to treat affective disorders in humans, but as environmental contaminant also affects inadvertently exposed fish in urban watersheds. In humans and fish, acute FLX treatment and exposure are linked to endocrine disruption, including effects on the reproductive and stress axes. Using the zebrafish model, we build on the recent finding that developmental FLX exposure reduced cortisol production across generations, to determine possible parental and/or life-stage-dependent (age and/or breeding experience) contributions to this phenotype. Specifically, we combined control and developmentally FLX-exposed animals of both sexes (F 0 ) into four distinct breeding groups mated at 5 and 9 months, and measured offspring (F 1 ) basal cortisol at 12 dpf. Basal cortisol was lower in F 1 descended from developmentally FLX-exposed F 0 females bred at 5, but not 9 months, revealing a maternal, life-stage dependent effect. To investigate potential molecular contributions to this phenotype, we profiled maternally deposited transcripts involved in endocrine stress axis development and regulation, epigenetic ( de novo DNA methyltransferases) and post-transcriptional (miRNA pathway components and specific miRNAs) regulation of gene expression in unfertilized eggs. Maternal FLX exposure resulted in decreased transcript abundance of glucocorticoid receptor, dnmt3 paralogues and miRNA pathway components in eggs collected at 5 months, and increased transcript abundance of miRNA pathway components at 9 months. Specific miRNAs predicted to target stress axis transcripts decreased ( miR-740 ) or increased ( miR-26 , miR-30d , miR-92a , miR-103 ) in eggs collected from FLX females at 5 months. Increased abundance of miRNA-30d and miRNA-92a persisted in eggs collected from FLX females at 9 months. Clustering and principal component analyses of egg transcript profiles separated eggs collected from FLX-females at 5 months from other groups, suggesting that oocyte molecular signatures, and miRNAs in particular, may serve as predictive tools for the offspring phenotype of reduced basal cortisol in response to maternal FLX exposure.

Effects of fluoxetine on fish: What do we know and where should we focus our efforts in the future?

“…Exposure to fluoxetine results in an anxiolytic effect, reducing the levels of cortisol detected in the body in response to stress. This cortisol levels reduction ability has been reported in the concentration range from 0.54 to 54 μg/L, from 15 min to 43 days (Abreu et al, 2015;Abreu et al, 2014;Fior et al, 2018;Martinez et al, 2019;Vera-Chang et al, 2018. However, increased levels or lack of effect have also been reported, in a range of concentrations from 10 to 5000 μg/L, from 1 h to 14 days (Abreu et al, 2016a;Giacomini et al, 2020;Marcon et al, 2016;Petrunich-Rutherford, 2019;Pittman and Hylton, 2015;Theodoridi et al, 2017).…”

“…The miRNAs play an important role in various biological processes such as cell proliferation, differentiation, apoptosis, and hematopoiesis (Chen et al, 2004;Dostie et al, 2003;Xu et al, 2003;Yao et al, 2019). In a study by Martinez et al (2019) with D. rerio embryos exposed to 54 μg/L fluoxetine, between 3 and 144 h, and then reared until the adult stage (± 5 months) in a clean medium, miRNAs in tissues and eggs were up-regulated when they were already in the adulthood. Alteration of egg stress axis transcript abundance was also reported.…”

“…It must also be highlighted that few studies studied effects of fluoxetine exposure on the offspring (generation F1 to F3), showing changes in cortisol levels, downregulation in the transcripts of the offspring stress axis, changes in the transcript abundance of the egg stress axis and the abundance of miRNAs in the eggs, and behavioral changes (reduced exploratory and locomotor behavior) (Martinez et al, 2019;Vera-Chang et al, 2018;Vera-Chang et al, 2019). These results raise some concern since these are based on short-term exposures (between 3 and 144 h) to low concentrations (0.54 and 54 μg/L) but show serious and harmful effects on the offspring.…”

Applications of the SR4G Transgenic Zebrafish Line for Biomonitoring of Stress-Disrupting Compounds: A Proof-of-Concept Study

“…As a proof-of-concept, two EDCs with known stress-disrupting properties, bisphenol A (BPA), a common plasticizer; and vinclozolin (VIN), a common fungicide (10, 24-28) were selected. Fluoxetine (FLX) was also studied because is it a widely used pharmaceutical with high stress-disrupting potential (29)(30)(31)(32). The glucocorticoid receptor (GR) agonist (hydrocortisone; CORT) was used as positive control for the stress response.…”

RNA-seq reveals potential gene biomarkers in fathead minnows (Pimephales promelas) for exposure to treated wastewater effluent

“…This includes disruption of normal reproductive and predator avoidance behaviors, 13 disruption of brain monoamine levels, 14 disruption of neurogenesis, 15,16 and transgenerational alteration of stress responses. 17,18 Consequently, these individual, sublethal impacts are implicated in or directly relate to the alteration of ecologically important behaviors that pertain to individual tness such as mating, predator escape responses, and feeding. These behavioral alterations could lead to population declines and ultimately have severe impacts on ecosystems.…”