Application of Molecular Biological Biomarkers to Endocrine Disruption Studies

In the natural environment, Endocrine Disrupting Chemicals (EDCs)may bring about great impact on the ecological environment and human health, and people have begun to understand the importance of studying the potential risks resulted from the pollution of EDCs. Molecular biomarkers provide an innovative technique for the screening and research of EDCs. This review included the methods of studying estrogenic biomarkers, thyroid hormone biomarkers, reproduction hormone biomarkers and latest progress of using EDCs biomarkers, aiming at ffective screening and detection of contaminants in EDCs. Abbreviations: Perchlorate; Perfluoroalkyl Catalase; SOD: Superoxide Dismutase; GPx: Glutathione Peroxidase; GSH: Glutathione; Malondialdehyde; T: Testosterone


Introduction
Endocrine Disrupting Chemicals (EDCs), also called environmental hormones, were first proposed in the book "Our Stolen Future" by a journalist Dianne Dumanoski [1]. EDCs are a group of xenobiotics greatly impacting the environment. As substances similar to endocrine hormones in living organisms, EDCs disrupt the normal secretion of hormones in bodies when entering animal and human bodies. This way, physiological disorders will occur, impacting the reproductive, nervous and endocrine systems of animals and humans or even causing carcinogenicity. Specifically, aquatic organisms are directly exposed to pollutants, and the endocrine interference impact on them is even greater [2][3][4]. EDCs feature various types, continuous production, diversified forms and bioaccumulation. In recent years, EDCs have become the most severe pollution issue after ozone depletion and global warming and attracted attention of many countries and international organizations [5].
Fish is often considered one of the model organisms for endocrine disruptor evaluation because endocrine disruptors may interfere with reproduction hormones in fish. First, sexual differentiation is unstable in fish, implying that during the sexual differentiation phase of this vertebrate, it may be disrupted by external hormones, or its sex may be even changed [6]. Second, aquatic ecosystem is under attack from various pollutants, and fish is directly exposed to industrial, agricultural and municipal wastewater and pollutants. Current evidence showed that longterm exposure to EDCs would affect reproduction and population of fishes [7]. Endocrine disruptors may affect important hormones or receptors by disrupting the endocrine pathway along the

Received: October 28, 2019
Hypothalamus-Pituitary-Gonad (HPG) axis of fish, which finally has an impact on animal reproduction. EDCs may also disrupt the Hypothalamic-Pituitary-Thyroid (HPT) axis of fish, interfere with thyroid hormone synthesis, transport and combination, destabilize the thyroid hormone environment, and are therefore harmful to the growth and development of fish. Environmental pollutants can affect the synthesis of steroid hormones and serve as an endocrine disruptor through a receptor independent pathway. Molecular biological technologies, such as -omics and transgenic techniques, provide a reliable approach to evaluate the threats from endocrine disruptors in environmental pollutants. They may also reveal the pollutant mechanism. In addition, specific genes in fish also help determine biomarkers as endocrine disruptors [8].
Biomarkers can be defined as an indicator for the level of exposure to and/or toxic effects of one or more chemical pollutants, which affect organisms at biological levels such as molecular, cell, and individual levels and cause measurable changes in tissues, biological fluids, or biochemical, cell, physiological, or behavioral features of organisms [9]. Some biomarkers can predict the effects of reproductive or other endocrine disruption based on the linkage between endocrine disruption and endpoints of molecular biological experiments. Therefore, biomarkers for EDCs now become a study focus after the establishment of highly efficient experimental methods reflecting the actual situation in organisms with a low cost and little or no damage. Biomarkers are widely applied to and play a significant role in endocrine disruption researches. Due to the diversified functions of EDCs, the types of biomarkers become increasingly abundant, with continuous emergence of a large number of new biomarkers.

Estrogenic Biomarkers
A distinctive feature of EDCs is their estrogenic effect. By emulating endogenous estrogens, EDCs lead to feminization of male individuals. Many synthetic compounds with different structures have estrogen or antiestrogen effects [10]. According to multiple studies, Vitellogenin (VTG) in adult fish may function as an ideal estrogenic biomarker. VTG is a precursor protein of egg yolk and is usually synthesized as a result of endogenous estrogen stimulation. VTG generally exists in the plasma of female fish. Though male fish also has VTG genes, VTG proteins cannot be autonomously synthesized in male fish in the absence of estrogen. However, stimulated by exogenous estrogen, VTG proteins may be synthesized in male fish. VTG is specific and sensitive to estrogen stimuli and has a high expression level in a number of organisms. Therefore, it can be used as an excellent estrogenic biomarker [11]. Recently, VTG expression was employed to evaluate the estrogenic effect of environmental chemicals. As compared to protein-based detection, VTG-based detection has a comparable sensitivity. Therefore, VTG can be employed as a biomarker to detect estrogenic effect at an early phase [l2,13]. At present, VTG genes in nearly 20 kinds of fish have been successfully cloned. Therefore, VTG may promote the applications of this biomarker in a wider range.
Estrogen plays a critical role in regulating the reproductive, developmental, and neuroendocrine systems of fish. In the study of the estrogenic effect, some other biomarkers have been discovered.
Estrogenic Receptors (ERs) and aromatase are essential for synthesis, secretion and physiological functions of estrogen. Estrogenic effect is produced when compounds simulate estrogen to combine with ERs and activate the ER genes. In bony fish, there are mainly two estrogenic receptor subtypes ERα and ERβ, which activate gene expression by binding to specific response elements [14,15].
In addition, estrogenic effect may be indicated by an increase in the level of free endogenous estrogens in plasma caused by the competitive binding of exogenous chemical hormones to globulin [16].
The antiestrogen effect implies that compounds have no or little estrogen activity but could perform competitive binding with ERs.
In addition, the antiestrogenic effect influence, the expression and activity of the cytochrome p450a19 enzyme (aromatase).

Thyroid Hormone Biomarkers
Another prominent feature of EDCs lies in the thyroid effect.
Thyroid hormone helps maintain the normal physiological conditions of vertebrates and is considered the most important factor in controlling their growth, development and behaviors. In bony fish, thyroid hormone also plays a vital role in regulating metabolism.
The synthesis and release of thyroid hormones are controlled by the HPT axis. Chemicals that disrupt one or more points in the HPT axis may affect the thyroid hormone function, and finally impact the growth and development of animals. Thyroid-disrupting chemicals may interfere with different regulation pathways, especially synthesis and metabolism (binding) of thyroid hormones, for example, synthesis through iodinated thyroglobulin or changing T4 into T3 with higher physiological activity. They may also disrupt plasma protein binding or combine with Thyroid Receptors (TRs) [22]. After combining with ligands, inhibitory factors activated proteins and TR homo-/hetero-dimers or retinoic X to induce the expression of target genes of thyroid response elements, such as phosphoenolpyruvate carboxykinase or 5'-diodinase [23]. In bony fish, there were two TR isomers (α and β) and splice variants [23]. The results helped explain the complicated chemical interactions and the molecular mechanisms of the two disruptors.

Reproduction Hormone Biomarkers
The HPG axis controls the reproduction of fish species among all vertebrates. During the fish spawning season, external factors such as light and temperature of water affect the gonad recovery and brain maturation. Signals from the brain control the Wei Zh et al. [31] studied the chronic reproductive toxicity and possible mechanism of Perfluoroalkyl Acids (PFAAs) after adult zebrafish (Danio rerio) was exposed to different concentrations Kyunghee J et al. [32] carried out a study on the impact of non-steroidal anti-inflammatory drugs (NSAIDs) on the PHG genes of zebrafish and their reproduction toxicity. This study found that ibuprofen and mefenamic acids significantly increased the concentrations of 17β-estradiol and testosterone in females, while decreased those of testosterone among male fish after the adult zebrafish was exposed to the drugs for 14 days. Significant upregulation of FSHβ, LHβ, FSHR and LHR was observed in females, whereas downregulation was observed in males exposed to NSAIDs.
After adult zebrafish pairs were exposed to ibuprofen for 21 days, and the egg production was significantly decreased at 1 µg/L ibuprofen, parental exposure resulted in delayed hatching even if they were transferred to clean water for hatching. The results demonstrated that ibuprofen could modulate hormone production and related gene transcription of the HPG axis in a sex-dependent way, which could cause adverse effects on the reproduction and development of offspring.
Qun-Fang Z et al. [33] conducted research on the reproductive toxicity of zebrafish under exposure to inorganic mercury (Hg).
After adult zebrafish was exposed to 0 (control), 15 and 30µg of Hg were changed. In females, although the ovarian 17β-estradiol (E2) content remained relatively stable, significant downregulation of LHR, GNRH2, GNRH3, LHR and EAR were observed. In males, Testosterone (T) levels in the testis significantly decreased after Hg exposure, accompanied by down-regulated expression of GNRH2, GNRH3, FSHβ and LHβ in the brain as well as FSHβ, LHβ, ARβ, CYP17 and CYP11b in the testis. Thus, our study indicated that waterborne inorganic Hg exposure altered sex hormone levels by disrupting the transcription of related HPG-axis genes, which could subsequently impair fish reproduction.

Conclusion
EDCs may bring about great impact on the ecological environment and human health, and people have begun to understand the importance of studying the potential risks resulted from the pollution of EDCs. It is important to perform researches on the toxicity of EDCs, especially the joint toxic effects caused by long-term exposure to endocrine disruptors at low dosage. The biological screening methods of EDCs, either in vivo or in vitro, require a high cost. Moreover, damages will be present in in-vivo biological tests, and significantly different results will occur in invitro tests when compared to in-vivo tests. Molecular biomarkers provide an innovative technique for the screening and research of EDCs. One of the advantages of this method is the low cost and effective reflection of actual conditions inside organisms. Another advantage lies in its sensitivity to the molecular endpoints in aromatase inhibitors, hormone levels and expressions of various genes. After fish is exposed for two to three weeks to chemicals at the concentration that can influence the reproduction, the molecular endpoints of the tests can be sensitively influenced. Using molecular endpoints is a relatively simple screening method for the study of endocrine disruption effects. Therefore, it is extremely important to use molecular biological technologies to establish a fast, reliable, specific and sensitive screening method for effective screening and detection of contaminants in EDCs.