The Effects of 17α-Ethinylestradiol (EE2) and Hydroxypropyl-β-Cyclodextrin (HPβCD) on the heart rate and metabolism of Embryonic Japanese Medaka (Oryzias latipes)

Abstract

Estrogen toxicity has been an area of priority in aquatic toxicology over the last 20 years. The toxicity of a known estrogen mimic, 17α-ethinylestradiol (EE2), has been attributed to classical estrogen signaling within target and non-target organisms. Recent evidence has indicated that a rapid, non-genomic, non-classical estrogen signaling pathway also exists via the G protein coupled estrogen receptor (GPER). GPER is expressed ubiquitously and has many biological functions, including cardiovascular and metabolic function. Understanding the role of GPER at environmentally relevant concentrations of estrogens could aid addressing many knowledge gaps associated with estrogen toxicity in aquatic environments. This thesis investigated the effects of EE2 on heart rate and metabolism, as well as EE2 uptake, and elimination in embryonic Japanese medaka (Oryzias latipes). Bradycardia (reduced heart rate) was observed in embryos exposed to 10 ng/L of EE2. It was demonstrated that these effects were the result of GPER activation and not estrogen receptor (ER) α and β. A mixture of EE2 and hydroxypropyl-β-cyclodextrin (HPβCD) was also investigated. HPβCD is a commonly used odour suppressant and excipient with the ability to include guest compounds. It was determined that the observed EE2 induced bradycardia was reduced with a 4:1 HPβCD : EE2 molar ratio. Uptake of 14C-EE2 followed a sigmoidal pattern, and chorion permeability increased as development progressed. Elimination of 14C-EE2, showed a pattern of exponential decay following exposure from 6-48 hours post fertilization. HPβCD did not impede uptake of 14C-EE2 across the chorion, suggesting that HPβCD may cross the chorion. Uptake of the HPβCD – EE2 mixture into the tissues of the developing embryo should be investigated. A conclusive link was not determined between EE2 induced bradycardia and embryonic oxygen consumption (metabolic rate). The absence of metabolic effects might be mitigated by cutaneous gas exchange by embryonic fish. This data suggests that embryonic heart rate may not be an ideal measure of metabolic rate in embryonic medaka. This thesis is valuable to the field of aquatic toxicology as it highlights GPER as a novel mechanism of action for EE2 toxicity as well as the role of HPβCD in mixture toxicity. This work and future research into the role of GPER will aid in the overall understanding of estrogen toxicity to fish.