Review ArticleAbstract
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: EDCs: Endocrine Disrupting Chemicals; HPT: Hypothalamic-Pituitary- Thyroid; HPG: Hypothalamus-Pituitary-Gonad; VTG: Vitellogenin; ER: Estrogenic Receptors; FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; TCDD: Tetrachlorodibenzo-Para-Dioxin; ZFL: Zebrafish Liver; TRs: Thyroid Receptors; SPs: Synthetic Pyrethroids; TH: Thyroid Hormone; PER: Perchlorate; GnRH: Gonadotropin Releasing Hormone; PFAAs: Perfluoroalkyl Acids; CAT: Catalase; SOD: Superoxide Dismutase; GPx: Glutathione Peroxidase; GSH: Glutathione; MDA: 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 [2-4]. 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 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).
Villeneuve et al. studied the impact of fadrozole on the
expression of genes related to reproduction in the HPG axis and
liver of fathead minnow. In the perspective of systems biology, they
established an evaluation system for the impact of pollutants on
the HPG and determined the association between gene expression
and experimental endpoints of reproductive biology. This method
aimed at the functions of multiple important organs instead of
only one organ such as the gonad, which helped understand
the influence and interaction between pollutants and the entire
endocrine system of fish [17]. Moreover, this approach depicted
the molecular function model and helped establish a relationship
reflecting the influence of molecular biological changes on organism
reproduction. Zhang et al. explored the impacts of prochloraz and
ketoconazole on the expression of reproduction-related genes in
the HPG axis and liver of medaka. They found a linear relationship
between the expressions of six genes in the liver and spawning
amounts. Therefore, gene expression amounts could be used to
speculate on the biological experiment endpoint of fish in the view
of ecology [18].
Dongmei et al. [19] studied the estrogen-related genes in the
HPG axis of the zebrafish embryo, or more specifically estrogenassociated
biomarkers including VTG1, VTG2, ERα, ERβ1, ERβ2,
CYP19a1a and CYP19a1b, based on which the joint effect of
compound estrogen involving cypermethrin, malathion and
prochloraz on zebrafish could be determined. The test results
demonstrated that within a period under a certain drug amount, the
estrogenic effect was effectively enhanced with binary pesticides
of cypermethrin and malathion or malathion and prochloraz when
compared with the treatment of a single chemical. In addition, the
expression amounts of ERα, ERβ1, ERβ2, CYP19b and CYP19a were
significantly changed.
Jinhua et al. [20] used molecular biomarkers such as estrogen
and thyroid hormone to study the endocrine disruption of acetochlor
at the early life stage of zebrafish. After zebrafish was exposed to
acetochlor at the concentrations of 50, 100 and 200 μg/L, the HPG/ HPT-associated genes in zebrafish, such as VTG1, ERβ1, CYP19a
and TRα, and many key genes in the apoptosis pathway, including
Bcl2, Bax, P53 and Cas8, underwent significant changes. The results
deomonstrated that acetochlor could induce oxidative stress and
apoptosis at the developmental stage of zebrafish and affect the
immune and endocrine systems of zebrafish. Yingying C. et al.
[21] reported the estrogenic effects of 2,3,7,8-Tetrachlorodibenzo-
Para-Dioxin (TCDD) on the gene expressions of the Zebrafish Liver
(ZFL) cell lines, and zebrafish embryo, larva, juvenile and adult
zebrafish livers, both independently and when combined with
Cd 2+. In ZFL cells, mRNA expression of VTG1 was significantly
suppressed by Cd2+ but was not affected by TCDD. In ZFL cells,
the expressions of ERα, ERβ1, ERβ2 and GPER were insignificantly
changed. They also conducted a study on the VTG1 promoter
deletion mutant and found no reaction in the presence of TCDD or
Cd2+. However, following co-transfection with a VTG1 promoterluciferase
construct to the ERα, ERβ1, ERβ2 and GPER expression
vectors, decreased luciferase activity was observed in the ERα cotransfection
group after treatment with Cd2+, suggesting that ERα
participated in VTG1 transcriptional regulation and was affected
by Cd2+. The regulation of these genes at the mRNA level varied in
male and female zebrafish livers at different developmental phases.
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].
It was difficult to identify insignificant impacts of thyroid hormone
on organisms in laboratories. However, molecular markers could
serve as an indicator and prediction signal to overcome the difficulty
in short-period experiments. Molecular biomarkers, which are
similar to estrogen regulation genes such as VTG, could be used as
potential sensitive biomarkers for thyroid hormones.
Wenqing et al. [24] studied the disruption of Synthetic
Pyrethroids (SPs) on the HPT axis of zebrafish embryo under acute
exposure conditions. SPs might damage the thyroid endocrine system of mammals. 1, 3 and 10 μg/L of bifenthrin or cyhalothrin
might influence the levels of T4 or T3. In addition, the genes such
as CRH, TSHβ, TTR, UGT1ab, Pax8, Dio2 and TRα in the HPT axis of
zebrafish were significantly upregulated as induced by bifenthrin.
Cyhalothrin could influence different test genes and significantly
induce the expressions of TTR, Pax8, Dio2 and TRα. However, the
Dio1 gene was greatly suppressed. At the atomic level, compared
with cyhalothrin, the combined protein of bifenthrin and TRα had
more powerful influence on the HPT signal conduction. According
to Shi et al. [25], PFOS had apparent development toxicity effect
on zebrafish embryo. The expressions of marker genes hhex and
pax8 for early thyroid development were significantly upregulated,
indicating possible thyroid development toxicity. The study on the
thyroid toxicity of PCB126 to salmon [26] revealed that the levels
of T4 in the salmon plasma and T4 glucosylation significantly rose,
resulting in swelling of the thyroid epithelial cells. As the herbicides
used with the largest amounts in China, acetochlor decreased the
Thyroid Hormone (TH) level according to the Crum’s study on
xenopus laevis, which impacted the thyroid functions. The author
explained the mechanism at the molecular level [27].
Xuesong et al. [28] studied the thyroid endocrine toxicity of
2,2’,4’,4-tetrabromodiphenyl ether (BDE-47) and Perchlorate (PER)
based on the TH-mediated pathway after zebrafish embryo was
exposed to them for 14 days. The test results indicated that when
compared with exposure to only BDE-47, combined exposure to
BDE-47 (10 μg/L) and PER (3.5 mg/L) could significantly upregulate
the genes NIS and Nkx2.1a that participated in TH synthesis,
which highly downregulated HPT axis (CRH and TSH) mediated by
expression of regulation-related genes. Compared to single BDE-47
exposure, combined exposure to BDE-47 and PER could greatly lift
the TG gene and protein levels, while significantly downregulated
the TTR gene and protein levels. In addition, exposure to the compounds
of BDE-47 and PER greatly decreased the T4 level, indicating
that the damage to the BDE-47 thyroid was increased by PER.
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
Gonadotropin Releasing Hormone (GnRH), and GnRH stimulates the
hypothalamus-pituitary, which secretes gonadotropin, including
Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH).
Then, LH and FSH, in return, control the production and secretion
of steroid hormones from the gonad. Hormones, including the
main hormones and sex inducing hormones of 17β-estradiol
and testosterone in female individuals, as well as testosterone
and 11-keto-testosterone in male individuals, initiate changes in
secondary sexual characteristics, in addition to the development
and maturation of sperm and eggs. In different physiological stages, reproduction hormones regulated the secretion of pituitary
gonadotropin hormones according to the positive or negative
feedback [29]. The HPG axis provided several potential modes,
which affected the synthesis and secretion of EDCs. By binding
to hormone receptors or changing the usability of endogenous
hormones, EDCs exerted their disruption function through the
mediation of steroid hormones or similar elements. Therefore,
biomarkers directly reflected the level of steroid hormones
and could function as a widely applied molecular indicator of
endocrine disruptors. VTG synthesis and secretion were induced
in livers by cyclic estrogen, and 17β-estradiol was dedicated to the
identification of exogenous estrogen [30].
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
(0.01, 0.1 and 1 mg/L) of PFAAs for 180 days. The disrupted
expression of genes, such as ERα, ERβ, FSHR, LHR, StAR and
17βHSD, indicated possible interference of PFNA on the HPGL axis
function and sex hormone synthesis. PFNA interfered with the
HPGL axis function and sex hormone synthesis by disturbing the
expression of genes in the HPGL axis, which led to estrogenic effects
such as significantly increased VTG content in males and increased
E2 levels in both genders. The results of this study provided a basis
for research on the potential risks of this ubiquitous and persistent
contaminant in aquatic ecosystems worldwide.
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
L (added as mercuric chloride, HgCl2) for 30 days, the activities and
mRNA levels of antioxidant enzymes (catalase (CAT), Superoxide
Dismutase (SOD) and Glutathione Peroxidase (GPx)) as well as
the contents of Glutathione (GSH) and Malondialdehyde (MDA)
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.
Acknowledgement
The research was supported by Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences and Zhejiang Province Major Bidding Project (Grant No. 2014C02002).
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