Superfund Research Program
Developmental Neurotoxicity of Xenoestrogens in Zebrafish
Synthetic chemicals and natural plant compounds originating outside the body that have a hormone-like activity in the body are referred to with a variety of terms: endocrine-disruptors, endocrine-modulators, environmental hormones, hormone-related toxicants, endocrine-active compounds, or hormonally active agents to name a few. In theory, all of the many hormones in the body have one or more environmental chemical mimic, but scientists have focused most attention on chemicals that are estrogen-like in their actions (known as xenoestrogens, environmental estrogens, or ecoestrogens). These compounds include drugs (birth control pills, cancer therapies, hormone replacement therapy), pesticides (DDT), industrial chemicals (PCBs), plasticizers (nonylphenol) and phytoestrogens (natural plant estrogens), and are widespread in the general environment and common contaminants at Superfund sites.
Endogenous estrogens, which originate within the organism, generally function by binding to specific receptor sites and turning on specific genes. Xenoestrogens can affect the hormonal system in a number of different ways, they can:
- bind to specific estrogen receptor sites, mimicking the endogenous hormone to evoke a hormone response
- bind to specific estrogen receptor sites, preventing the hormone from binding with the site thus blocking or inhibiting the proper hormone response
- bind to other receptors and create a novel reaction or interfere indirectly with normal hormonal action
- alter production and breakdown of hormone receptors and natural hormones, which changes hormonal blood concentrations and endocrine responses.
The result is inappropriate regulation of genes including incorrect timing or too much or too little cellular activity. Because exposure to xenoestrogens in early development (embryo or fetus) can produce changes that are not evident until much later in life it is often difficult to establish that exposure has occurred or to evaluate the relationships between exposure and impact on an organism. Scientists at Boston University are working to demonstrate the utility of the zebrafish (Danio rerio) as a whole animal, in vitro system for detecting neural estrogen effect in embryos. A second goal of their research is to investigate the hypothesis that xenoestrogens act as neurodevelopmental toxicants by virtue of their ability to disrupt the normal timing or level of gene expression during early central nervous system development.
Estrogens are known to have organizational effects in the developing central nervous system and are believed to be important in neurodevelopment and lifelong processes of neuroplasticity. Certain neural actions of estrogen are dependent on the chemical transformation of circulating androgen to estrogen in the brain itself. This estrogen formation in the brain and other estrogen synthesizing tissues is catalyzed by the enzyme cytochrome P450 aromatase (P450arom). Interestingly, the gene that encodes P450arom (and estrogen production) is itself a target of estrogen. For example, seasonal increases in estrogen levels associated with normal reproductive cycles have been shown to upregulate brain P450arom levels (and brain estrogen production), suggesting a possible mechanism for xenoestrogen effects on the nervous system.
Teleost fish are ideal experimental models for this study because they have exaggerated levels of neural P450arom and throughout life retain a remarkable potential for neurogenesis and neuroregeneration. In addition, teleost fish have two separate and distinct P450arom isoforms in nonneural (P450aromA) and neural tissues (P450aromB). This allows researchers to identify brain-specific estrogen-mediated activities. Because messenger RNA (mRNA) directs protein synthesis, the Boston University scientists were able to evaluate P450arom activity by quantifying P450arom mRNA levels relative to untreated controls. They demonstrated that the P450aromB isoform is transcribed from the earliest stages of embryogenesis and is estrogen-inducible.
To test the applicability of P450aromB mRNA as a marker of xenoestrogen effects in the central nervous system, the Boston University researchers exposed zebrafish embryos to:
- 17ß-Estradiol (E2), a natural estrogen hormone in humans and many animals
- diethylstilbestrol (DES), a potent synthetic estrogen that was used as a human medication to help maintain pregnancy and in the treatment of prostate cancer decades ago but was removed from these uses due to human carcinogenicity issues
- bisphenol A (BPA), a relatively weak estrogen used in industrial processes to make polycarbonate plastic resins, epoxy resins, and other products.
They determined that the lowest level of E2 tested induced a nearly fourfold increase in P450aromB mRNA. Both DES and BPA were able to mimic E2, inducing upregulation of P450aromB mRNA in zebrafish embryos. Interestingly, DES was found to be more potent than E2, while BPA failed to increase P450aromB mRNA to the level induced by E2. In each trial, the ovarian P450aromA mRNA was unaffected. These data suggest that P450aromB mRNA levels in zebrafish embryos could be used for screening known and suspected xenoestrogens in aquatic environments, and that environmental exposure to xenoestrogens could alter the timing and amount of estrogen biosynthesis in the developing central nervous system.
Interference with hormonal function is particularly dangerous to the developing embryo and neonate. During early developmental stages, alteration of brain or body development is irreversible and potentially life threatening. To examine the possible developmental effects of exposure to excess estrogen, the Boston University scientists incubated embryos with E2, DES, BPA and androgen for specific time periods. They found that treatment with 10 m M E2 between 2 and 72 hours post-fertilization increased mortality and induced pericardial enlargement and the curved tail phenotype characteristic of mutants with defects of early central nervous system development. DES produced similar effects - at concentrations lower than the E2 treatment. BPA treatment had much less impact than E2, while androgen was completely inactive. The scientists note that the curved tail effects are likely not mediated by changes in P450aromB levels, but may be the result of general estrogen toxicity acting via nongenomic mechanisms of estrogen action.
Embryos treated with 10 mM E2 between 2 and 72 hours post-fertilization (hpf) hatched later than controls and embryos treated with lower doses of E2. To determine the critical developmental period of estrogen sensitivity, embryos exposed to 10 m M E2 between 2 and 72 hpf were compared to those exposed only at 2 to 24, 24 to 48, and 48 to 72 hpf. The Boston University scientists determined that the critical period of estrogen effect on survival and morphological defects is 2 to 24 hpf. They found that longer duration of the E2 treatment did not increase mortality or rate of pericardial enlargement or curved tails. However, the effect of 10 m M E2 estrogen on delayed hatching required the full 2 to 72 hpf exposure period.
The proper development and functioning of the central nervous system is critical to the overall development and functioning of the organism. Correct developmental programming requires genes and processes to be turned on and off in a strict spatial and temporal order. These studies demonstrate that xenoestrogens can interfere with hormonal function in very early stages of embryogenesis, which in turn can impact gene expression and cell processes in the rapidly developing nervous system. Although the research at Boston University identified critical periods of estrogen effects on survival, hatching, and morphological defects, the consequences of inappropriate estrogen exposure for the development and lifelong functioning of the nervous system are still under study. Because estrogen-like chemicals are common contaminants at Superfund sites, and can be transmitted to the developing mammalian embryo, fetus and newborn through maternal blood or breast milk, quantifiable, mechanism-based molecular indicators are needed to recognize their presence at polluted sites and to characterize the biological effects of these chemicals on animal and human populations. This research represents the first step towards the development and validation of a whole animal, in vitro screening system using the brain form of P450arom as a marker of neuroactive xenoestrogen effect.
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To learn more about this research, please refer to the following sources:
- Kishida M, Callard GV. 2001. Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development. Endocrinology 142(2):740-750. doi:10.1210/endo.142.2.7928 PMID:11159846
- Kishida M, McLellan M, Miranda JA, Callard GV. 2001. Estrogen and xenoestrogens upregulate the brain aromatase isoform (P450aromB) and perturb markers of early development in zebrafish (Danio rerio). Comp Biochem Physiol 129(2-3):261-268. PMID:11399458
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