The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting chemical (EDC) of concern due to its persistent nature and widespread presence in the aquatic environment. In mammals, effects of developmental EDC exposure on reproduction and behavior not only persist to adulthood after discontinued exposure, but are also inherited by several consecutive unexposed generations. The results presented in this thesis demonstrate that non-reproductive behavior in fish is highly sensitive to the influence of EE2 during development and the effects do not appear to be restored after a long recovery period in clean water. We have shown that exposure to low doses of EE2 during development results in increased anxiety in two fish species (zebrafish and guppy) and their offspring. We have also demonstrated that the effects of EE2 on anxiety are apparent in both sexes and are transgenerationally transmitted to two consecutive generations of unexposed offspring in the guppy. In order to investigate the possible biological mechanisms of the observed persistent effects on non-reproductive behavior, we also performed an RNA sequencing analysis of the whole-brain transcriptome in developmentally exposed zebrafish after remediation in clean water until adulthood. Differential expression of 33 genes in males and 62 genes in females were observed as a result of EE2 exposure, with only one gene affected in both sexes. Functional analysis revealed cholesterol biosynthesis and circadian rhythm to be the top two affected pathways in males and females, respectively. Both pathways have previously been implicated in anxiety behavior and represent possible candidates connecting the transcriptome alterations to the observed behavioral phenotype. The study represents an initial survey of the fish brain transcriptome by means of RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

Selective serotonin reuptake inhibitors (SSRIs), like fluoxetine, are widely used to treat depressive disorders during pregnancy. These antidepressants reach water reservoirs through sewage treatment facilities and expose the aquatic vertebrates, including fish. It has been shown that early-life exposure to fluoxetine could disrupt the normal function of the stress axis by decreasing the level of circulating glucocorticoids in humans, rodents, and teleosts. Our lab recently showed that early life exposure to fluoxetine resulted in transgenerational hyporcortisolism and altered exploratory behaviour in adult male zebrafish and their descendant male adults for at least three generations. In the current study, we used a stress-responsive transgenic zebrafish line (SR4G) that expresses green fluorescence protein (eGFP) under the control of six consecutive glucocorticoid response elements. The effects of developmental exposure to fluoxetine on the transcriptional profiles of genes in the larval head and male adult telencephalon and hypothalamus were analysed using high throughput RNA sequencing. We also assessed the potential of eGFP mRNA to evaluate blunted stress response as an alternative to cortisol immunoassay measurements. The effects of bisphenol A, vinclozolin and fluoxetine were ytested in the SR4G line. Developmental exposure to fluoxetine resulted in a life-long dysregulation of pathways involved in nervous system development, stress response, and lipid metabolism in both larvae and adult zebrafish. Numerous differentially expressed genes in zebrafish are orthologous to genes in Homo sapiens linked the development of the major depressive disorder and epigenetics regulation and include bdnf, trkb, npas4, per1, per2, dnmt3a, adarb1, adaeb2, hdac4, hdac5, hdac8, and atf2. It is suggested that the dysregulation of the primary transcription regulators of circadian rhythm (clocka) and stress response (nr3c1), amongst others, were the potential drivers of the observed life-long effects. Furthermore, we report on a significant positive linear correlation between cortisol levels and eGFP mRNA levels in SR4G transgenic zebrafish larvae (R2> 0.9). Random forest and logistic regression models trained by eGFP mRNA levels both correctly predicted the blunted stress response. The negative predictive value (NPV) for both models was 100%. Models based on the mRNA levels of 11 genes associated with neurogenesis, stress response and depression resulted in a similar 100% NPV. These findings provide evidence for a life-long effect of developmental exposure to fluoxetine. This study also provides a proof-of-concept for an in vivo biomonitoring assay to screen chemicals for their stress-disrupting potential.