Bisphenol A (BPA) is a chemical incorporated in plastics and resins used for food and beverage containers that has been shown to have estrogenic activity. The fact that BPA possess this activity should not be surprising as it was originally explored for use as a pharmaceutical estrogen. Exposure to BPA has been associated with adverse reproductive and developmental effects in wildlife and laboratory animal models. There are also associations between exposure in humans and adverse health effects, although some of these findings are controversial. The mechanism(s) of action of BPA are well researched, however there is no definitive explanation for the frequently reported discrepancies between in vitro and in vivo studies. Metabolic activation of BPA in vivo has been suggested as a possible reason for this discrepancy in estrogenic potency. As public awareness of the possible health effects of BPA increases manufacturers have increasingly started to use replacement chemicals as monomers in materials that can be labelled as BPA-free. However there is still little information on the estrogenic potency of these structurally similar bisphenol chemicals or how they may affect health outcomes, as observed with BPA. The studies conducted in this thesis therefore aimed to investigate the tissue targets, molecular mechanisms and health effects of BPA, its related chemicals Bisphenol S (BPS), Bisphenol F (BPF) and Bisphenol AF (BPAF) and the BPA metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). For this work, a novel ERE transgenic (ERE-TG) zebrafish, that expresses green fluorescent protein (GFP) in response to activation of ERE was employed. These fish can be applied to identify body targets of environmental estrogens in real time with high sensitivity and specificity.
BPA, BPF, BPS and BPAF were shown to all preferentially target the heart in ERE-TG zebrafish and GFP induction occurred first in the heart out of the different responding body tissues. The response to BPA was shown to be dependent on the classical estrogen receptor (ER) signalling pathway. However concentrations necessary to induce this response varied for the different bisphenols, with the rank order of potency of BPAF>BPA=BPF>BPS. Bioconcentration factors of the bisphenols were 4.5, 17.8, 5.3 and 0.067 for exposures to 1000 µg BPA/l, 1000 µg BPF/l, 100 µg BPAF/l and 50000 µg BPS/l respectively. These data indicate bioavailability is an important consideration in the differing estrogenic potencies of the different bisphenols. The toxicities of the different bisphenols on early life stage zebrafish followed a similar rank potency order as for the estrogenic activity (BPAF>BPA>BPF>BPS). Specific morphological abnormalities were observed for the different bisphenolic chemical treatments in the toxicity assessments, possibly suggesting that they may act through different ways in inducing their toxic effects. It is recognised that the toxicities for the bisphenolic chemicals were observed at concentrations several orders of magnitude higher than those measured in most aquatic environments and thus the threat they pose to wildlife health might be considered as relatively low, except in circumstances where short but high exposures may occur from accidental release into the environment.
The BPA metabolite MBP was found to be up to 1000-fold more potent than the parent compound as an estrogen in ERE-TG fish. The heart was a key target tissue for MBP, as observed for the other bisphenolic compounds. The atrioventricular valves and bulbus arteriosus were identified as the primary targets within the heart. MBP was not measured in zebrafish embryos exposed to BPA and whether this is produced as a metabolite in zebrafish is still not known. Morpholino knockdown of specific ER subtypes indicated that esr1 is a major pathway for the estrogenic response to BPA in the heart during early life stages of zebrafish. Video capture and analysis was used to assess the cardiovascular health of zebrafish exposed to BPA and it was found that at very high exposure concentrations (2500 µg/l) BPA could induce an unstable atrial:ventricular beat ration in 5 dpf larvae and reduced heart beat rate in 14 dpf.
In the final study of this thesis transcriptomic profiling was conducted on hearts extracted from 96 hpf ERE-TG zebrafish larvae exposed to BPA. The findings demonstrated that BPA, at an exposure concentration of 150 µg/l caused a down-regulation of a number of genes associated with ion transport and cell-to-cell communication, functions that are essential in maintaining a regular and consistent heart rate. These effect mechanisms may help to explain the effects on the heart seen at the higher BPA exposure concentrations in the previous chapter, although this would need more extensive work to draw any such associations with good confidence
Overall, the findings presented in this thesis have provided a body of evidence to show that all of the bisphenolic chemicals tested possess estrogenic activity and as such have the potential for health effects in wildlife and also to humans. It is also the case however that currently in most ambient environments concentrations of these bisphenolic chemicals are far below those that could induce adverse health outcomes. The work in this thesis re-enforces the importance of understanding metabolic activation of chemicals in vivo. It furthermore illustrates the power of transgenic fish and an integrated approach for gaining greater insight into potential health effects of chemicals.NER
