Tissue Targets, Molecular Mechanisms and Health Effects of Bisphenolic Chemicals in Zebrafish

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

Acute Toxicity, Teratogenic, and Estrogenic Effects of Bisphenol A and Its Alternative Replacements Bisphenol S, Bisphenol F, and Bisphenol AF in Zebrafish Embryo-Larvae

International audienceBisphenol A (BPA), a chemical incorporated into plastics and resins, has estrogenic activity and is associated with adverse health effects in humans and wildlife. Similarly structured BPA analogues are widely used but far less is known about their potential toxicity or estrogenic activity in vivo. We undertook the first comprehensive analysis on the toxicity and teratogenic effects of the bisphenols BPA, BPS, BPF, and BPAF in zebrafish embryo-larvae and an assessment on their estrogenic mechanisms in an estrogen-responsive transgenic fish Tg(EREGal4ff)(UASGFP). The rank order for toxicity was BPAF > BPA > BPF > BPS. Developmental deformities for larval exposures included cardiac edema, spinal malformation, and craniofacial deformities and there were distinct differences in the effects and potencies between the different bisphenol chemicals. These effects, however, occurred only at concentrations between 1.0 and 200 mg/L which exceed those in most environments. All bisphenol compounds induced estrogenic responses in Tg(EREGal4ff)(UASGFP) zebrafish that were inhibited by coexposure with ICI 182 780, demonstrating an estrogen receptor dependent mechanism. Target tissues included the heart, liver, somite muscle, fins, and corpuscles of Stannius. The rank order for estrogenicity was BPAF > BPA = BPF > BPS. Bioconcentration factors were 4.5, 17.8, 5.3, and 0.067 for exposure concentrations of 1.0, 1.0, 0.10, and 50 mg/L for BPA, BPF, BPAF, and BPS, respectively. We thus show that these BPA alternatives induce similar toxic and estrogenic effects to BPA and that BPAF is more potent than BPA, further highlighting health concerns regarding the use of BPA alternatives

Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol A (BPA) and Its Metabolite 4‑Methyl-2,4-bis(<i>p</i>‑hydroxyphenyl)pent-1-ene (MBP) in Zebrafish

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis­(4-hydroxyphenyl)­pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the <i>bulbus arteriosus</i> were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures