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Mechanisms of action of agrochemicals acting as endocrine disrupting chemicals
Journal Pre-proof Mechanisms of action of agrochemicals acting as endocrine disrupting chemicals Genoa R. Warner, Vasiliki E. Mourikes, Alison M. Neff, Emily Brehm, Jodi A. Flaws PII:
S0303-7207(19)30382-X
DOI:
https://doi.org/10.1016/j.mce.2019.110680
Reference:
MCE 110680
To appear in:
Molecular and Cellular Endocrinology
Received Date: 1 October 2019 Revised Date:
6 December 2019
Accepted Date: 10 December 2019
Please cite this article as: Warner, G.R., Mourikes, V.E., Neff, A.M., Brehm, E., Flaws, J.A., Mechanisms of action of agrochemicals acting as endocrine disrupting chemicals, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2019.110680. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Abstract Submission MCE-D-19-00600 Mechanisms of Action of Agrochemicals Acting as Endocrine Disrupting Chemicals
Agrochemicals represent a significant class of endocrine disrupting chemicals that humans and animals around the world are exposed to constantly. Agrochemicals can act as endocrine disrupting chemicals through a variety of mechanisms.
Recent studies have shown that
several mechanisms of action involve the ability of agrochemicals to mimic the interaction of endogenous hormones with nuclear receptors such as estrogen receptors, androgen receptors, peroxisome proliferator activated receptors, the aryl hydrocarbon receptor, and thyroid hormone receptors. Further, studies indicate that agrochemicals can exert toxicity through non-nuclear receptor-mediated mechanisms of action. Such non-genomic mechanisms of action include interference with peptide, steroid, or amino acid hormone response, synthesis and degradation as well as epigenetic changes (DNA methylation and histone modifications).
This review
summarizes the major mechanisms of action by which agrochemicals target the endocrine system.
1 2 3 4 5
Review
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Mechanisms of Action of Agrochemicals Acting as Endocrine Disrupting Chemicals
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Special Issue on “Health effects of agrochemicals acting as endocrine disrupters”
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Genoa R. Warner, Vasiliki E. Mourikes, Alison M. Neff, Emily Brehm, Jodi A. Flaws
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Department of Comparative Bioscience, University of Illinois at Urbana-Champaign, Urbana, IL 61802
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Corresponding author
15 16 17 18 19
Jodi A Flaws [email protected] 2001 S. Lincoln Ave Urbana, IL 61802
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22 23
Introduction In Silent Spring in 1962, Rachel Carson recognized that chemicals in the environment,
24
particularly the pesticide dichlorodiphenyltrichloroethane (DDT) and other agrochemicals, were
25
impacting reproduction and development in wildlife and had the potential to harm humans (Carson,
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1962). Although she did not know it at the time, Rachel Carson was describing the effects of endocrine
27
disrupting chemicals (EDCs), compounds that interfere with the action of hormones in the body (Gore et
28
al., 2015). Over the past half century, scientists have identified the mechanisms through which DDT
29
causes the softening of eggshells and decreases in bird populations described in Silent Spring as well as
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numerous other mechanisms through which environmental chemicals can interfere with the complex
31
operations of the endocrine system.
32
In this review, we summarize endocrine disruptor mechanisms of action with an emphasis on
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agrochemicals that act as EDCs. The endocrine system operates through complex and finely tuned
34
hormone signaling pathways. As such, EDCs exhibit numerous modes of action. The most well-known
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mechanisms of EDC action involve mimicking the interaction of endogenous hormones with nuclear
36
receptors, but evidence supporting non-nuclear receptor-mediated mechanisms of action will also be
37
discussed herein.
38
The Endocrine System
39
The endocrine system controls reproduction, development, growth, metabolism, tissue and brain
40
function, and other physiological functions in the body. Endocrine glands distributed throughout the
41
body, including the brain, thyroid, mammary glands, cardiovascular system, and reproductive organs,
42
produce and release hormones. Understanding the complexities of hormone signaling provides vital
43
context to the wide range of EDC mechanisms.
44 45
The canonical pathway of hormone signaling involves the binding of a hormone to its corresponding nuclear receptor(s). Ligand binding induces structural changes in the receptor that lead to
46
dimerization, exposure of co-factor binding sites, and DNA binding. Genomic binding may occur directly
47
to response elements in the genome (Figure 1A) or through transcription factors (Figure 1B). Hormones
48
may also function through non-genomic rapid signaling via secondary messengers (Figure 1C).
49
Exogenous chemicals, including pharmaceuticals and EDCs, can mimic hormones as ligands via any of
50
these three mechanisms as well as repress transcription by recruiting co-repressors (Figure 1D) (Heldring
51
and Pike, 2007).
52 53
Figure 1: Model of the canonical hormone signaling via nuclear receptors (NR). Hormones (grey
54
triangles) act as ligands for nuclear receptors which can bind directly to DNA (A) or indirectly via
55
transcription factors (TF) (B) to regulate gene expression. Hormones may also signal through non-
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genomic pathways by interacting with membrane nuclear receptors or cytoplasmic receptors to initiate
57
signaling cascades via secondary messengers (SM) that lead to rapid physiological effects without
58
altering gene expression (C). Hormone mimicking chemicals may operate through any of the mechanisms
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shown in A-C as well as recruit co-repressors to block gene transcription (D). Adapted from Helding and
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Pike, 2007.
61
Extremely low concentrations of hormones are present in the body, on the order of pg–ng/mL for
62
estradiol, testosterone, and thyroid hormone (Vandenberg et al., 2012). Furthermore, only small
63
percentages of many circulating hormones, including steroid and thyroid hormones, are bioavailable; the
64
majority are bound to carrier proteins. The endocrine system is tuned to respond to minor changes in
65
bioavailable hormone concentrations. Signaling at these extremely low concentrations is facilitated by the
66
high affinity of hormones for their receptors as well as non-linear relationships between hormone
67
concentration, receptor occupancy, and biological effect (Welshons et al., 2003). Thus, a change in
68
hormone concentrations at low doses has a stronger effect than the same magnitude of change at a higher
69
dose. For a full discussion of this, see Vandenberg et al., 2012. In the context of understanding endocrine
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disruptor mechanisms, this means that studying low doses of EDCs (below those typically used in
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toxicological testing) is required to identify mechanisms, and that the mechanisms may change at
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different doses. This is evident in non-monotonic dose response curves (NMDRCs) observed in
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toxicological studies of EDCs, in which the sign of the curve changes (i.e. U or inverted U) as the dose
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changes. NMDRCs are established for hormonal pharmaceuticals such as tamoxifen yet they continue to
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be challenged in the context of EDCs (Vandenberg et al., 2012).
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EDC Mechanisms of Action
77
Endocrine disrupting chemicals can bind to receptors to mimic endogenous hormones, but they
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also act by altering hormone signaling in a variety of other ways. EDCs may interact with multiple
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receptors, including non-nuclear receptors, as agonists, in which they facilitate genomic interactions, or as
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antagonists, in which they cause a conformational change to the receptor to block action. They may also
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trigger non-genomic signaling that is independent of nuclear receptors. Importantly, EDCs can interfere
82
with endogenous hormone synthesis and degradation to alter hormone levels. Recent studies have also
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identified how EDCs can follow an epigenetic mode of action by altering genomic methylation and
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histone modifications.
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Nuclear Receptors
86
Nuclear receptors are ligand dependent transcription factors that bind hormones and exert long
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term control of their target cell phenotype. This is in contrast to membrane receptors which elicit faster,
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short term effects on their respective cells. Nuclear receptors play a crucial physiologic role in
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development, organ homeostasis, metabolism, immune function, and reproduction (Balaguer et al., 2019).
90
Hormones are the endogenous ligands of most of the major classes of nuclear receptors, but EDCs may
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also bind nuclear receptors. Nuclear receptors can act as repressors or activators of gene transcription
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depending on ligand binding status, the identity of the ligand itself, and available coregulators.
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Nuclear receptors are composed of several conserved structural domains, including a DNA
94
binding domain and a ligand binding domain (Weikum et al., 2018). The DNA binding domain is
95
responsible for interacting with the genome and typically interacts with specific sequences called DNA
96
response elements, whereas the ligand binding domain contains the binding pocket for small molecules.
97
Unliganded nuclear receptors can be found in the nucleus or cytoplasm and may bind to target genes,
98
resulting in recruitment of corepressors. Following endogenous ligand binding, conformational changes to
99
nuclear receptors disrupt corepressors leading to dimerization and interaction with DNA and co-
100
activators. Upon binding to DNA or interacting with tethering co-factors, nuclear receptors further
101
activate co-factors to facilitate transcription.
102
Endocrine disrupting chemicals can interact with nuclear receptors directly and elicit strong
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biological consequences (Gore et al., 2015). Agricultural chemicals, including herbicides, insecticides,
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rodenticides, and fungicides, are prime examples of synthetic endocrine disrupting molecules exogenous
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to normal eukaryotic biology that interact with nuclear receptors (Beischlag et al., 2008). Table 1
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provides examples of the biological effects of select agrochemicals that act through nuclear receptors.
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Table 1: Agrochemical effects mediated through nuclear receptors Chemical atrazine
dibutyltin (DBT)
DDT
DDE dieldrin & aldrin
fenarimol
lindane (γ-HCH)
Mode of Action
Effect
Reference
AhR mediated
Nephrotoxicity
(Zhang et al., 2019)
PPARγ activation
Androgenic antagonist: inhibits dihydrotestosterone (DHT) binding to AR
ESR1 activation leading to altered expression of Pgr, Ccnd1, Cyp19a1 AR antagonist: inhibits DHT binding to AR AR antagonist AR antagonist leading to decrease in prostate binding protein, ornithin decarboxylase, and insulinlike-growth factor 1 mRNA levels Androgenic antagonist: inhibits DHT binding to AR
linuron
AR antagonist
methoxychlor
ESR1 and ESR2 agonist
methoxychlor procymidone
ESR1 and ESR2 agonist AR antagonist
propiconazole & tebuconazole 2,3,7,8tetrachlorodibenzodioxin (TCDD)
Pregnane X receptor (PXR) activation
2,3,7,8tetrachlorodibenzodioxin (TCDD) tributyltin (TBT)
AhR activation
AhR activation during in utero exposure
AhR activation
Increased expression of adipogenic genes, promoted adipogenic differentiation, increased lipid accumulation, decreased glucose tolerance, increased circulating leptin levels in males Decreased fertility and cryptorchidism. Reduction of testicular weight, decreased in number and motility of spermatozoa in epididymis, loss of gametes in lumen of seminiferous tubules, decreased testosterone production by testes, increased luteinizing hormone and follicle stimulating hormone Hormonal carcinogenesis in uterus and ovaries Decreased fertility and cryptorchidism Reproductive performance affected
(ChamorroGarcía et al., 2018)
(Kelce et al., 1995; Sakly, 2001)
(Kalinina et al., 2017) (Kelce et al., 1995) (Danzo, 1997)
Reduced weight of ventral prostate, seminal vesicles, and bulbourethral glands
(Vinggaard et al., 2005)
Altered testis histology
(Danzo, 1997)
Disruption of reproductive tract development, reduction of accessory gland weight, reduction of epidydimal weight, increased serum estradiol and luteinizing hormone Premature nuclear expression of ER gene in neonatal uterine epithelium Inhibition of folliculogenesis and stimulation of anti-Müllerian hormone production in the ovary In males: shortened anogenital distance, hypospadias, reduced weight and altered histology of prostate, permanent nipples, ectopic undescended testes Hepatocellular hypertrophy and hepatocellular steatosis Cleft palate Downregulation of uterine and hepatic ESR1 results in repressed estradiol function UGT1 induction resulting in decreased serum T4 concentrations and compensatory excretion of thyroid
(Lambright, 2000)
(Eroschenko et al., 1996) (Uzumcu et al., 2006) (Ostby et al., 1999) (Knebel et al., 2019) (Abbott et al., 1996) (Romkes et al., 1987) (Nishimura et al., 2002)
stimulating hormone PPARγ and retinoid X receptor (RXR) activation PPARγ induction and Esr1 repression tributyltin (TBT) vinclozolin AR antagonist
In utero exposure results in increase lipid accumulation in adipose tissue, liver, and testis Impaired metabolic functions on liver and pancreas via lipid accumulation in white adipose tissue and hepatic inflammation Hypospadias, undescended testes, delayed puberty, transgenerational prostate disease
(Grün et al., 2006) (Bertuloso et al., 2015) (Monosson et al., 1999; Christiansen et al., 2008; Anway, 2015)
108 109
The structural similarities between EDCs and endogenous hormones form the basis for nuclear
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receptor-based mechanisms of endocrine disruption. Both classes of molecules are generally hydrophobic,
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lipid-soluble small molecules. In addition, many of the most worrisome EDCs have structural elements
112
such as hydroxyl groups in the proper position to interact with amino acid residues in the ligand binding
113
pocket in the same manner as endogenous hormones. For example, the methoxychlor metabolite 2,2-bis-
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(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) contains both hydroxyl groups and hydrophobic
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aromatic regions that allow it to bind to estrogen receptors, whereas the pyrethroid deltamethrin shares a
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diphenyl ether structural moiety with thyroid hormones (Delfosse et al., 2015; Du et al., 2010) (Figure 2).
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Once EDCs bind a receptor, they either activate the hormone receptor, amplifying physiological hormonal
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activity, or antagonize endogenous hormone action to block activity. Due to the structural similarities
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between EDCs and endogenous hormones as exemplified in Figure 2, EDCs are typically investigated as
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competitive binders in the ligand binding domain of the receptor. In addition, EDCs which are not
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structurally similar to hormones can still directly affect nuclear receptors by stimulation or inhibition of
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receptor expression. This leads to imbalances in endocrine homeostasis through modification of hormone
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receptor turnover/availability.
124 125
Figure 2: Structural similarities between endogenous hormones and agrochemicals. Top: Estradiol
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and the methoxychlor metabolite HPTE share phenolic structure (blue) that facilitates the binding of
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HPTE to human estrogen receptor 1 (ESR1) (Delfosse et al., 2015). The second hydroxyl group of HPTE
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does not interact in the ESR1 binding pocket in the same way as the second hydroxyl on estradiol.
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Bottom: The thyroid hormone triiodothyronine (T3) and pyrethroid deltamethrin share diphenyl ether
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functional groups (green) that facilitate binding to thyroid receptors (Du et al., 2010).
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In vitro assays are valuable tools for investigating the ability of EDCs to directly interact with
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receptors and alter transcription processes. One commonly used assay is a recombinant receptor and
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reporter gene assay in which a luciferase reporter gene is associated with a nuclear receptor response
134
element such that binding of a ligand to the receptor triggers transcription and luciferase activity (Legler
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et al., 1999). Although these assays are specific, responsive, and quick, they do not measure binding
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affinity nor identify which step of the nuclear receptor activation process is disrupted (Xiang et al., 2017;
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Judson et al., 2018). High throughput screening of nuclear receptor hormone agonists and antagonists is a
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top priority of regulatory agencies. In the US, the Tox21 program high throughput screening program
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utilizes multiple in vitro and in silico assays to assess ligand binding, dimerization, DNA binding, RNA
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transcription, and other steps in the activation process (Judson et al., 2018). However useful in vitro and
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silico models are for predicting EDC interaction with nuclear receptors, these models do not integrate
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multiple mechanisms of endocrine disruption that can occur in a whole animals. For this reason, in vivo
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testing is also necessary to understand mechanisms that may occur simultaneously in multiple organ
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systems to extrapolate to human and environmental health (Gore et al., 2015).
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Estrogen Receptors (ESR)
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Estrogen receptor alpha and estrogen receptor beta (ESR2) have unique and overlapping
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physiological roles that are highly tissue and cell type dependent. Both receptors are expressed in the
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brain, lungs, uterus, ovaries, breast, heart, and intestines. ESR1 is predominantly expressed in hepatocytes
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of the liver and the hippocampus, whereas ESR2 is predominantly expressed in the prostate, vagina, and
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cerebellum (Taylor and Al-Azzawi, 2000). ESR1 and ESR2 have quite similar binding pockets with
151
subtle differences in amino acids at the ligand binding domain that explain the ligand selectivity between
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the two types (Kuiper et al., 1998). Both ESR1 and ESR2 regulate gene expression in response to
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estrogen exposure via ligand dependent or ligand independent mechanisms, and each subtype can mediate
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unique responses to ligands.
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Inappropriate ESR signaling can lead to increased risk of hormone-dependent cancer, impaired
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fertility, abnormal fetal growth and development, and altered metabolism in white adipose tissue (Zhao et
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al., 2008). Many EDCs display estrogenic activity and interfere with normal estrogen signaling mediated
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by ESR1 and ESR2. A notable characteristic of ESRs that makes them susceptible to direct interaction
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with EDCs is their large binding pockets and broad specificity for ligands. The binding pockets are much
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larger than the estrogen molecule itself (Pike et al., 1999; Brzozowski et al., 1997). Although ESR1 and
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ESR2 have similar affinities for estrogen, EDCs and other exogenous ligands may display higher affinity
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for one subtype over the other. For example, the o,p' isomer of DDT, which is the most estrogenic isomer
163
of DDT, has a higher relative binding affinity for ESR2 than ESR1 (Kuiper et al., 1998).
164
Organochlorine pesticides, including methoxychlor and DDT, are notorious estrogenic
165
agrochemicals. They bind both ESR1 and ESR2 (Kuiper et al., 1998) and elicit ER binding to DNA both
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directly and through tethering as described above. DDT adversely affects the female reproductive tract by
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stimulating uterine proliferation and impairing normal follicle development (Tiemann, 2008). Although
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DDT was banned in the 1970s, it is persistent in the environment and accumulates in adipose tissue,
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making it a relevant threat to public health today. DDT and its dechlorination metabolite
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dichlorodiphenyldichloroethylene (DDE) have been detected in human adipose tissue around the world
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many years after its use was terminated (Turusov et al., 2002).
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Methoxychlor is another potent endocrine disruptor which stimulates uterotrophic activity and
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impairs overall fertility in rat models (Cummings, 1997). Methoxychlor itself has low binding affinities
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for ER. However, its major metabolite HPTE is a well-studied agonist for both ESR1 and ESR2 and is
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likely the responsible agent for the endocrine disrupting effects of methoxychlor (Gaido et al., 2000).
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HPTE acts as an agonist for ESR1 and antagonist for ESR2 (Gaido et al., 2000). ESR1 knockout mice do
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not respond to HPTE treatment, indicating the effects of HPTE on gene regulation in the mouse uterus are
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dependent on ESR1. HPTE may also mediate effects through non-classical ER signaling mechanisms
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similar to estrogens. HPTE and estrogen treatment led to similar gene expression profiles in uteri from
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mice expressing an ESR1 mutant deficient in DNA binding, which limits ESR1 mediated gene regulation
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to the pathway in which ESR1 tethers to DNA through transcription factors like AP-1 and Sp1 (Hewitt
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and Korach, 2011).
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Pyrethroids also exhibit estrogenic and antiestrogenic activity, with variable results observed in
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tests of different chemicals in vitro, suggesting that individual screening of members of a class of
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pesticides is necessary to identify endocrine disrupting activity (Saillenfait et al., 2016). In addition to in
186
vitro screening, whole-organism bioassays have been used to detect the estrogenic activity of EDCs. In
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transgenic medaka expressing green fluorescent protein, the herbicide linuron elicited anti-estrogenic
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activity, whereas fenoxycarb, a less-studied insecticide, showed no effect on estrogen, androgen, or
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thyroid signaling (Spirhanzlova et al., 2017). Recent studies have also investigated the estrogenic
190
properties of mixtures of pesticides. An in vitro analysis using multiple screening methods of individual
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pesticides and mixtures on ESR1 and ESR2 found additive effects of the mixtures (Seeger et al., 2016),
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suggesting the need for further experiments on mixtures, which are more representative of human
193
exposure.
194
Androgen Receptor
195
The androgen receptor (AR) is a ligand dependent nuclear transcription factor responsible for
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male fetal development, secondary sex characteristics at puberty, and maintenance of spermatogenesis.
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Beyond sex differentiation, the AR plays a critical role in development and maintenance of the
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reproductive, musculoskeletal, cardiovascular, immune, neural, and haemopoietic systems (Rana et al.,
199
2014). Consequences of dysfunctional AR include infertility (Nenonen et al., 2011), delayed puberty
200
onset (Mouritsen et al., 2013), and cryptorchidism (Davis-Dao et al., 2012). AR signaling is also involved
201
in the development of tumors in the prostate, bladder, liver, kidney and lung.
202
The fungicides vinclozolin (Gray et al., 1994), procymidone (Hosokawa et al., 1993), and
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prochloraz (Vinggaard et al., 2002) target the AR and their phenotypic consequences have been well
204
documented. Five triazole fungicides, tebuconazole, uniconazole, hexaconazole, peneconazole, and
205
bitertanol showed anti-androgenic activity toward human AR both before and after metabolism mediated
206
by human liver microsomes (Lv et al., 2017). Many insecticides and their metabolites have also been
207
shown to inhibit androgen receptor dependent transcriptional activity. These include multiple isomers of
208
DDT, DDE, and dichlorodiphenyldichloroethane (DDD) as well as methoxychlor and HPTE (Maness et
209
al., 1998). Urea based herbicides such as linuron have also been identified as endocrine disruptors via AR
210
antagonism (Lambright, 2000; Spirhanzlova et al., 2017). Agrochemicals have also been studied for their
211
effects against AR as mixtures. Various combinations of androgen antagonists have been shown to disrupt
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male reproductive development in a cumulative and additive matter, regardless of their individual modes
213
of action, and differences in tissue selectivity (Wilson et al., 2008).
214 215
Peroxisome Proliferator Activated Receptor
The peroxisome proliferator activated receptor (PPAR) is a ligand activated nuclear receptor that
216
participates in energy combustion via stimulation of lipid catabolism and energy storage via stimulation
217
of adipogenesis (Neschen et al., 2007). Lipophilic hormones, monounsaturated fatty acids,
218
polyunsaturated fatty acids, and eicosanoids are all endogenous ligands of PPAR (Ayisi et al., 2018).
219
Three subtypes of PPAR, PPARα, PPARβ, and PPARγ exist, with differing tissue distribution, ligand
220
specificity, physiologic role, and mechanism of action. Once bound to their endogenous ligands, PPARs
221
can induce the expression of genes and enzymes involved in lipid metabolism through both genomic and
222
non-genomic mechanisms.
223
In a study of the PPARα and PPARγ activity of 200 pesticides using in vitro reporter gene assays,
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only three pesticides, doclofop-methyl, pyrethrins, and imazalil, exhibited PPAR agonist activity, which
225
was further confirmed in vitro (Takeuchi et al., 2006). Due to its primary physiologic role as a regulator
226
of adipogenesis and lipolysis, PPARγ is a major target of agricultural chemicals that act as obesogens.
227
Tributyltin (TBT), an antifouling agent, alters PPAR-mediated differentiation of adipocytes and promotes
228
adipogenesis in liver and adipose tissue (Maradonna and Carnevali, 2018). Exposure to dibutyltin (DBT),
229
the major metabolite of TBT in the body, binds PPARγ to accelerate adipogenesis in both human and
230
mouse mesenchymal stem cells. Interestingly, human cells were shown to be significantly more
231
responsive to DBT than mouse cells (Chamorro-García et al., 2018). The fungicide triflumizole has also
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been shown to activate PPARγ to promote adipogenesis, acting as an obesogen in vivo (Li et al., 2012).
233
Aryl Hydrocarbon Receptor (AhR) Complex
234
The aryl hydrocarbon receptor complex includes the aryl hydrocarbon receptor (AhR) and the
235
aryl hydrocarbon receptor nuclear translocator (ARNT). Although it is a basic region-helix/loop.helix
236
(bHLH) protein and not a nuclear receptor, the AhR is a ligand dependent transcription factor with similar
237
structure and function to nuclear receptors (Nebert, 2017). Following ligand binding, the AhR forms a
238
heterodimer with ARNT that binds to DNA at xenobiotic response elements (Figure 3). For many years
239
prior to the discovery of endogenous AhR ligands, the receptor’s most notable role was metabolism of
240
exogenous, synthetic chemicals. The Ahr is activated by exogenous chemicals, and relays environmental
241
signals to the cell (Elferinks et al., 1990). Thus, many mechanisms through which the AhR mediate toxic
242
effects in the body are well studied. Much of what is known today regarding mechanisms of AhR
243
mediated modulation of gene expression has been studied in the context of dioxin metabolism. Recently,
244
endogenous ligands of AhR have been identified, whereas the receptor’s physiological roles in cell
245
proliferation and differentiation, immune response, inflammation, and regulation of circadian rhythm are
246
less understood (Nebert, 2017).
247 248
Figure 3: Mechanism of AhR signaling. Synthetic chemicals such as dioxin (green triangles) activate
249
AhR by binding to the receptor and translocating to the nucleus, where the complex dimerizes with the
250
aryl hydrocarbon receptor nuclear translocator (ARNT) and controls transcription by interacting with
251
xenobiotic response elements (XRE) in DNA. Transcriptional regulation by dioxin and other EDCs leads
252
to an upregulation of xenobiotic metabolism to create toxic metabolites that impact the immune system,
253
neurological function, reproduction, and carcinogenesis.
254
2,3,7,8-Tetrachlorodibenzodioxin (TCDD, also known as dioxin) is one of the most widely
255
studied AhR ligands. The AhR has been shown to modulate the activity of other transcription factors
256
including ESRs and ARs (Ohtake et al., 2011). In the presence of estrogens and androgens, the AhR-
257
ARNT heterodimer downregulates transcriptional events that would otherwise be upregulated in the
258
presence of these hormones. Dioxin bound AhR acts as a substrate specific adaptor component that
259
targets estrogen and androgen bound receptors for degradation by the cullin 4B ubiquitin ligase complex
260
(Ohtake et al., 2007). The opposite effect on transcription has been observed when AhR is activated in the
261
absence of estrogen and androgen hormones. The AhR-ARNT complex physically associates with
262
unliganded estrogen and androgen receptors bringing transcriptional co-activators to the promotors of
263
each of these receptors (Ohtake et al., 2003). Hexachlorobenzene, a banned but persistent dioxin-like
264
fungicide, also activates AhR to trigger both genomic and non-genomic effects (van Birgelen, 1998; Miret
265
et al., 2019).
266
Reporter gene assays of 23 pesticides and insecticides in human and rat cell lines indicated AhR
267
agonistic effects by iprodione, chlorpyrifos, and prochlorax, whereas five additional pesticides exhibited
268
mixed activity in the two cell lines (Long et al., 2003). Another reporter gene assay study identified
269
eleven pesticides that agonize AhR function, while two inhibited AhR activity (Ghisari et al., 2015).
270
Pyrethroids have structural elements reminiscent of dioxins, suggesting potential AhR binding activity
271
(Brander et al., 2016); cypermethrin has been shown to antagonize TCDD-induced AhR transactivation
272
(Ghisari et al., 2015). The fungicide propiconazole also activates AhR (Knebel et al., 2018). This has
273
been confirmed in silico, in vitro, and in vivo, wherein AhR was activated in a luciferase reporter gene
274
assay in a human cell line and further increased mRNA and enzyme expression of genes controlled by
275
AhR in the livers of rats treated with propiconazole via diet (Knebel et al., 2018).
276
Thyroid Receptors
277
Thyroid receptors (TR) are another class of ligand-dependent transcription factors. Studies show
278
that four active isoforms of thyroid receptors (TRα1, TRβ1, TRβ2, and TRβ3) bind the endogenous
279
thyroid hormones triiodothyronine (T3) and thyroxine (T4) (Zoeller, 2012). TRα and TRβ isoforms are
280
differentially expressed in tissues throughout the body. Isoform distribution is particularly important
281
during fetal brain development when thyroid hormone signaling is vital for normal development. Thyroid
282
receptors are also important for physiological control of metabolism and the cardiovascular system
283
(Zoeller, 2012).
284
Extensive evidence in humans and animals indicates that pesticides including chlorpyrifos, DDT,
285
and methoxychlor can disrupt thyroid signaling and neurodevelopment (Boas et al., 2012). However,
286
most evidence on pesticides implicates thyroid hormone synthesis, bioavailability, and metabolism for
287
these effects (Ghassabian and Trasande, 2018). Pesticides generally do not have a high degree of
288
structural similarity to thyroid hormones and thus have been less investigated as TR binders compared to
289
environmental chemicals such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers
290
(PBDEs) that have been shown bind directly to TR (Ghassabian and Trasande, 2018; Zoeller, 2007).
291
However, in vitro reporter gene assays have indicated the ability of multiple pesticides to bind to TR. Ten
292
pyrethroids or pyrethroid metabolites have demonstrated antagonistic activity against TR (Du et al.,
293
2010). In an in vitro reporter assay analysis of the binding of 21 pesticides to human TR, 13 bound
294
directly to TR, with 5 exhibiting agonistic activity, including procymidone, imidacloprid, mancozeb, and
295
atrazine, whereas 11 were antagonistic and 4 exhibited both agonistic and antagonistic activity (Xiang et
296
al., 2017).
297
Non-genomic Signaling
298
While genomic mechanisms of endocrine disruption involve activation of nuclear receptors that
299
translocate to the nucleus to modulate gene expression, EDCs also exhibit non-genomic mechanisms of
300
action. These actions are characterized as rapid effects that do not directly or initially influence gene
301
expression (Figure 1C) (Lösel and Wehling, 2003). A non-genomic effect can be observed in a much
302
shorter time frame (seconds to minutes of exposure to experimental chemical) than genomic effects that
303
are constrained by transcription and translation. These effects are also typically unaffected by
304
actinomycin D or cycloheximide treatment, which inhibit transcription and translation, respectively.
305
Many non-genomic actions are initiated at the level of the cell membrane through interactions
306
with membrane-associated receptors. The fungicides prochloraz and vinclozolin have been shown to
307
directly target the membrane androgen receptor ZIP9, a member of the solute carrier protein family, to
308
block testosterone-driven zinc influx and apoptosis in PC3 prostate cancer cells (Thomas and Dong,
309
2019). Another study has demonstrated that the herbicide atrazine and pesticide trans-nonachlor target the
310
epidermal growth factor receptor (EGFR) and block receptor activation and autophosphorylation
311
(Hardesty et al., 2018). Further protein-ligand docking simulations suggest that trans-nonachlor acts as a
312
competitive antagonist to the EGFR, whereas atrazine blocks the tyrosine kinase activity of the receptor
313
(Hardesty et al., 2018). The insecticide cypermethrin can also inhibit EGFR activity and downstream
314
MAPK activation by interfering with non-classical testosterone signaling in sertoli cells, leading to
315
reduced cell viability and proliferation (Wang et al., 2019).
316
Activation of membrane receptors triggers signal transduction cascades to regulate cellular
317
responses like changes in gene expression, proliferation, and metabolism. The mitogen activated protein
318
kinase pathway (MAPK) is a kinase cascasde activated downstream of membrane receptors for growth
319
factors and cytokines that regulate cell growth, survival, and differentiation (Morrison, 2012). Several
320
studies have shown MAPK pathway activation to be critical in mediating the effects of agricultural
321
chemicals like nonylphenol, atrazine, and cypermethrin on Sertoli cell viability, testosterone production in
322
Leydig cells, and gonadotropin production in cultured gonadotropes, respectively (Choi et al., 2014;
323
Pogrmic-Majkic et al., 2016; F. Li et al., 2018). One study observed reduced membrane associated
324
connexin43 expression and impaired inter- and intracellular signaling in liver progenitor cells treated with
325
either methyoxylchlor or vinclonzin (Babica et al., 2016). This effect was dependent on p38 MAPK
326
activity as well as activation of protein kinase C.
327
The phosphoinositide-3-kinase–protein kinase B/AKT (PI3K/AKT) pathway is another target of
328
agricultural chemicals. The PI3K/AKT pathway is often triggered upstream by activation of membrane
329
receptors targeted by growth factors and cytokines, and plays a critical role in cell proliferation, survival,
330
and metabolism (Hemmings and Restuccia, 2012). Current studies have demonstrated the suppression of
331
this signaling pathway by nonylphenol and ziram in the testes of exposed rats, resulting in the generation
332
of reactive oxygen species and increased apoptosis (Huang et al., 2016; Xie et al., 2018).
333
Another common target of agricultural chemicals is the cyclic adenosine 3′,5′-monophosphate
334
(cAMP) pathway. The second messenger cAMP accumulates in the cell through activation of adenylyl
335
cyclase by membrane associated G-protein coupled receptors (Sassone-Corsi, 2012). In turn, cAMP
336
activates protein kinase A (PKA) to regulate cell metabolism, ion transport, and gene expression
337
(Sassone-Corsi, 2012). Atrazine has been shown to increase cAMP levels in Leydig cells by inhibiting the
338
cAMP specific phosphodiesterase, an enzyme that catalyzes the hydrolysis of cAMP to the inactive 5-
339
AMP (Karmaus and Zacharewski, 2015). Excessive accumulation of cAMP by atrazine increases PKA
340
activity to modulate testosterone production in Leydig cells (Samardzija et al., 2016; Pogrmic-Majkic et
341
al., 2016; Karmaus and Zacharewski, 2015). Other chemicals, like methyoxychlor and its metabolite
342
HTPE, have been shown to inhibit follicle stimulating hormone (FSH) stimulated cAMP production,
343
resulting in reduced estrogen production (Harvey et al., 2015). Another study demonstrated that exposure
344
to a mixture containing organochlorine insecticides impaired cAMP signaling, leading to decreased
345
cAMP mediated post-translational processing of the steroidogenic acute regulatory protein to its active
346
form and reduced steroidogenesis in cultured Leydig cells (Enangue Njembele et al., 2014).
347
EDCs can also influence cell function by targeting membrane associated ion-channels to mediate
348
rapid non-genomic responses. These channels open to form a pore, allowing the passive diffusion of
349
specific ions into the cell that will mediate numerous cellular functions, including the control of electrical
350
excitability and the exocytosis of secreted proteins (Jentsch et al., 2004). Insecticides cypermethrin and
351
DDE as well as glyphosate-based herbicides have been shown to target membrane bound voltage gated
352
calcium channels and/or intracellular ryanodine receptors and increase intracellular calcium
353
concentrations (Tavares et al., 2013; De Liz Oliveira Cavalli et al., 2013; Ye et al., 2017; F. Li et al.,
354
2018). Perturbation in calcium homeostasis by agricultural chemicals has been linked to several
355
physiological effects, including reduced acrosomal integrity and mobility in sperm, increased oxidative
356
damage and decreased viability in Leydig cells, and increased glucose-stimulated insulin secretion from
357
pancreatic β-cells through generation of reactive oxygen species (Tavares et al., 2013, 2015; De Liz
358
Oliveira Cavalli et al., 2013; Chen et al., 2017). Another study demonstrated that cypermethrin induced
359
calcium influx triggered rapid activation of MAPK signaling, leading to increased gonadotropin secretion
360
from cultured pituitary cells (F. Li et al., 2018). Cypermethrin has also been shown to target voltage gated
361
sodium channels in the hypothalamus to increase gonadotropin-releasing hormone pulse frequency (Ye et
362
al., 2017).
363
The non-genomic effects of endocrine disrupting chemicals are not fully understood, and even
364
less is known about those driven by agricultural EDCs. This is partly due to the duration of treatment.
365
Many studies using agrochemicals involve long term treatment periods spanning days to weeks, which
366
make it difficult to discern the rapid non-genomic actions from the slower genomic actions. Use of shorter
367
exposure periods in in vitro culture systems and the inclusion of actinomycin D and cycloheximide will
368
greatly increase our understanding of the non-genomic mechanisms of agricultural EDCs.
369
Hormone Synthesis and Degradation
370
Hormones in the body can be characterized into three main groups depending on their major
371
components: peptide/protein hormones, steroid hormones, and amino acid analogue hormones.
372
Peptide/protein hormones are the most abundant in the body and include gonadotropin-releasing
373
hormone, growth hormone, FSH, luteinizing hormone (LH), and thyroid stimulating hormone (TSH).
374
Important steroid hormones include estrogens, progesterone, and testosterone. Lastly, amino acid
375
analogues include iodothyronines such as thyroxine (T4) and 3,5,3’-triiodothyronine (T3) and amines
376
such as dopamine and serotonin. The synthesis and degradation of peptide/protein, steroid, and thyroid
377
hormones can be negatively affected by EDCs, including agrochemicals. Interfering with hormone action
378
may cause negative downstream effects on reproductive and non-reproductive health.
379
Peptide/protein hormones
380 381
Peptide and protein hormones are composed of short and long amino acid chains, respectively. Synthesis occurs in the nucleus and cytoplasm of secretory cells via gene transcription, translation into the
382
peptide chain, and finally post-translational modifications. After hormones are synthesized and packaged
383
into secretory granules, neural and hormonal signals cause their secretion into extracellular space
384
(Malandrino and Smith, 2018). Some peptide and protein hormones are secreted in pulsatile patterns that
385
may have rhythmic changes that contribute to feedback mechanisms, such as the hypothalamic-pituitary-
386
gonadotropin (HPG) axis, to control other hormone production in the body. Most peptide/protein
387
hormones are soluble in aqueous solvents and do not need carrier proteins for transport throughout the
388
blood stream. Therefore, they are susceptible to rapid protease degradation, leading to a short half-life and
389
duration of action (Malandrino and Smith, 2018). TSH, a glycoprotein hormone that stimulates the
390
production of T4, is susceptible to EDC influence. Perinatal exposure in rats to a glyphosate-based
391
herbicide resulted in decreased TSH levels, decreased gene expression of deiodinases and transporters,
392
and altered other genes regulated by thyroid hormones or involved in thyroid hormone metabolism in
393
male offspring (de Souza et al., 2017). Epidemiological studies of agricultural workers have shown
394
associations between pesticide exposure and levels of FSH and LH (Recio et al., 2005; Cremonese et al.,
395
2017). Extensive evidence in animal studies also indicates that chlorpyrifos treatment alters levels of FSH
396
and LH, in addition to steroid hormones (Li et al., 2019).
397
Steroid Hormones
398
Most steroid hormone synthesis takes place in the adrenal glands, ovary, testes, placenta, and
399
adipose tissue. The synthesis of steroid hormones is known as steroidogenesis. Cholesterol is the pre-
400
cursor for all steroid hormones, and it is transferred across the mitochondrial membrane by steroidogenic
401
acute regulatory protein (StAR). Cholesterol is then converted to pregnenolone in a process that is
402
common to all steroidogenic pathways (Miller and Auchus, 2011). After the production of pregnenolone,
403
the pathway varies depending on the hormone being produced; pregnenolone may undergo various
404
biotransformations to form aldosterone and cortisol in the adrenal gland or progesterone, estradiol, and
405
testosterone in the gonads (Acconcia and Marino, 2018; Auchus, 2014) (Figure 4). Most steroids are
406
hydrophobic in nature so hormones and their precursors can leave steroidogenic cells easily and are not
407
stored (Acconcia and Marino, 2018). After a biological response occurs, hormone secretion ceases which
408
contributes to feedback loops in the body, including the HPG axis and the hypothalamic-pituitary-adrenal
409
(HPA) axis (Acconcia and Marino, 2018). Carrier proteins transport steroid hormones throughout the
410
body and control the proportions of bound and unbound hormones (Leung and Farwell, 2018). Steroid
411
hormones are eliminated through enzymatic reactions followed by transport across the cell membrane.
412
Many of these reactions occur in the liver and include hydroxylation, conjugation, and reduction-
413
oxidation (You, 2004). After metabolites reach circulation, they are readily excreted in both urine and bile
414
as unconjugated or conjugated metabolites, but some may remain in circulation by binding to serum
415
proteins (You, 2004).
416 417
Figure 4: Steroidogenesis. Cholesterol is imported by steroidogenic acute regulatory protein (StAR) and
418
transformed into pregnenolone, which is further converted to adrenal and gonadal hormones.
419
Agrochemical exposure causes detrimental effects on the process of steroidogenesis that occurs in
420
the testis, ovary, and adrenal gland. These effects have been observed in both in vivo and in vitro
421
experimental designs. Alteration of steroidogenesis is a widely studied mode of action of EDCs; thus,
422
Table 2 provides examples of recent studies on the impacts of agrochemicals on steroid hormones.
423
Considering the literature, some common targets of agrochemical EDCs emerge. Multiple chemicals have
424
been shown to affect the synthesis of cholesterol, which is the pre-cursor for all steroid hormones. In the
425
adrenal glands, agrochemicals negatively affect the hormones, genes, and proteins involved in the
426
synthesis of steroid hormones. In the ovary, agrochemicals disrupt the production of estradiol by altering
427
the levels of enzymes and pre-cursor hormones involved in steroidogenesis in both in vivo and in vitro
428
studies. In the testes, agrochemicals impair the synthesis of testosterone, leading to detrimental effects on
429
reproduction. Table 2: Agrochemical effects on steroid hormone synthesis, metabolism, and concentrations Chemical
Mode of Action
Effect
Reference
2,4-D
Decreased serum testosterone levels, testis testosterone levels, Cyp17a1 levels, and total cholesterol levels in Leydig cells
Decreased pregnancy rate and number of pups
(Harada et al., 2016)
Decreased luminal spermatozoa
(Zhang et al., 2017)
Decreased testis, seminal vesicle, and prostate weight
(Pogrmic et al., 2009)
Altered dilation of seminiferous tubules and decreased size of Leydig cells
(Victor-Costa et al., 2010)
Altered steroidogenesis and aromatase activity
(Tinfo et al., 2011)
Decreased percent cell viability in Leydig cells
(Abarikwu et al., 2011)
Decreased levels of estradiol, but increased levels of progesterone in granulosa cells
Impaired reproductive efficiency
(Basini et al., 2012)
Altered levels of steroidogenic enzymes and increased levels of progesterone and testosterone in Leydig cells
Altered steroidogenesis
(Forgacs et al., 2013)
Increased levels of estrogen and androgens in adrenal cells
Interference with androgen synthesis
(Háhn et al., 2016)
Reduced levels of 3β-HSD expression
Decreased testis weight and heterogeneous testis morphology Decreased female fertility
(MartinsSantos et al., 2017) (PogrmicMajkic et al., 2018) (Ji et al., 2019)
Decreased serum testosterone levels atrazine
Decreased steroidogenic enzymes in Leydig cells, decreased serum levels of dihydrotestosterone and testosterone, decreased transport of cholesterol to mitochondria Decreased testosterone levels and increased estradiol levels, decreased protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) expression Increased estradiol and progesterone levels in granulosa cells, increased estradiol, estrone, and progesterone levels in adrenal cells Altered levels of steroidogenic enzymes
Reduced levels of steroidogenic enzymes
cypermethrin
Altered levels of steroidogenic enzymes, increased levels of estrogen, cortisol, and aldosterone in adrenal cells
Altered the steroidogenic pathway
Increased levels of steroidogenic enzymes, increased levels of luteinizing hormone, follicle-stimulating hormone, and testosterone
Accelerated onset of puberty in males
Altered levels of steroidogenic enzymes, decreased levels of testosterone, increased levels of estradiol in vivo and in vitro
Altered testis weight over time, decreased germ cells, impaired spermatogenesis and steroidogenesis Decreased testis weight, sperm count, sperm motility, sperm viability, sperm production, and inhibited spermatogenesis Impaired spermatogenesis and decreased sperm count
endosulfan
Decreased steroidogenic enzyme and testosterone levels
fenvalerate
Decreased expression of steroidogenic enzymes and decreased serum and testicular testosterone Decreased levels of corticosterone, testosterone, cholesterol, but increased weight of adrenal glands Altered levels of steroidogenic enzymes, decreased progesterone levels, but increased estradiol levels Increased levels of cholesterol but decreased levels of steroidogenic enzymes in the ovary, decreased levels of cholesterol, but increased levels of steroidogenic enzymes in the adrenal glands, and deceased serum levels of LH, FSH, estradiol, and progesterone Decreased levels of steroidogenic enzymes and levels of testosterone, FSH, and LH
glyphosate and glyphosatebased herbicides lambdacyhalothrin
mancozeb
Decreased levels of steroidogenic enzymes and levels of testosterone
Decreased testosterone and FSH levels
methoxychlor
ziram
Altered levels of steroidogenic enzymes, decreased testosterone levels, increased estradiol levels Decreased levels of steroidogenic enzymes and testosterone Decreased levels of most steroidogenic enzymes and decreased levels of estradiol, testosterone, androstenedione, and progesterone Decreased expression of steroidogenic enzymes, levels of testosterone, and levels of FSH Decreased steroidogenic enzyme, testosterone, and FSH levels
430 431
Amino Acid Hormones
Disrupted function of HPA axis
Decreased ovary weight, altered ovarian histology, and sex ratio of pups Altered ovarian structural degenerations and follicular maturation
Delayed Leydig cell development Decreased testis, epididymis, seminal vesicle, vas deferens, and prostate weight, decreased sperm parameters Increased abnormal sperm morphology and sperm viability Decreased testis weight and disrupted spermatogenesis
(Ye et al., 2017) (Huang and Li, 2014)
(Aly and Khafagy, 2014)
(Zhang et al., 2010) (Pandey and Rudraiah, 2015) (Ren et al., 2018) (R. Ghosh et al., 2018)
(H. Li et al., 2018) (Girish and Reddy, 2018)
(Elsharkawy et al., 2019) (Du et al., 2014)
Decreased testis weight, cauda sperm count, and sperm motility Decreased antral follicle growth
(Aly and Azhar, 2013)
Decreased epididymis and testis weight
(Guo et al., 2017)
Disrupted Leydig cell development
(Xie et al., 2018)
(Basavarajappa et al., 2012)
432
Amino acid analogue hormones develop from amino acids. Specifically, the amines are derived
433
from tyrosine and are secreted from both the thyroid and adrenal medulla (Koibuchi, 2018). The thyroid
434
gland synthesizes the endogenous thyroid hormones T4 and T3, which require dietary iodine. Thyroid
435
hormones are produced extracellularly in the follicular lumen (Koibuchi, 2018). The lumen is filled with
436
the glycoprotein thyroglobulin, which is synthesized within the epithelial cells and secreted into the
437
lumen (Koibuchi, 2018). Iodine is oxidized by the enzyme thyroperoxidase and then binds to tyrosine
438
residues in the thyroglobulin, resulting in the formation of iodotyrosines (Koibuchi, 2018; Kopp et al.,
439
2008). The iodotyrosines are coupled by thyroperoxidase to form T4, which is further converted into T3
440
in target tissues by deiodinases. Thyroid hormones are transported via carrier proteins including thyroid
441
binding globulin and transthyretin. The levels of T4 and T3 are regulated by deiodinases in peripheral
442
tissues and metabolism in the liver (Boas et al., 2012).
443
Although pesticides are generally poor structural mimics for thyroid hormones, they have been
444
shown to exert significant effects on thyroid hormone synthesis, concentrations, and carrier proteins.
445
Nonylphenol, a now-banned surfactant previously used in pesticide formulations, inhibits thioperoxides
446
necessary for the synthesis of T4. (Schmutzler et al., 2004) The triazole herbicide amitrole has been
447
shown to increase the transcription of thyroglobulin gene, while decreasing the uptake of iodide (Pan
448
Hongmei et al., 2011). Further, DDT has been shown to down-regulate the iodine-accumulation function
449
of follicular thyrocytes by suppressing sodium/iodide symporter synthesis and disrupting regular thyroid
450
function in male rats (Yaglova and Yaglov, 2015). Multiple human epidemiological studies have
451
indicated an association between disrupted thyroid hormone levels and pesticide use (Cremonese et al.,
452
2017; Recio et al., 2005; Piccoli et al., 2016; Blanco-Muñoz et al., 2016; Lacasaña et al., 2010).
453
Malathion, nonylphenol, ioxynil, and pentachlorophenol exposure decreased T3 binding to Japanese quail
454
transthyretin (TTR), one of the carrier proteins, with no observed interaction with the ligand binding
455
domain of thyroid receptors (Ishihara, Nishiyama, et al., 2003). Dicofol was shown to alter T3 binding to
456
bullfrog TTR in a biphasic manner, with low doses of dicofol increasing binding and high doses
457
inhibiting binding (Ishihara, Sawatsubashi, et al., 2003). Recently, enhanced binding of T4 to human TTR
458
has been demonstrated in the presence of organophosphate triesters, which are typically used as flame
459
retardants, but have similar structures to organophosphate pesticides (Hill et al., 2018).
460
Epigenetics
461
Epigenetics encompasses changes in gene expression without changing DNA sequences. The
462
epigenome, which consists of all chemical and structural marks that control the accessibly of the genome,
463
is highly sensitive to environmental conditions including nutrition and stress as well as chemical
464
exposures. Changes to the epigenome can be heritable and can manifest in health impacts and disease
465
long after the exposure has ended, even multiple generations after the exposure occurred (Rattan and
466
Flaws, 2019). Observed transgenerational effects of environmental chemicals were one of the first
467
indications that EDCs could alter the epigenome. For example, vinclozolin and methoxylchlor exposure
468
during pregnancy have been shown to decrease fertility in male descendants for up to four generations
469
(Anway et al., 2005). The effects of these chemicals on germ cell genomes make these outcomes
470
heritable. Development is also a particularly sensitive window of exposure due to the extensive genetic
471
programming that occurs in this time period. Exposures during periods of development can alter the
472
epigenome to increase susceptibility to disease later in life, after the exposure has ended. This forms the
473
mechanistic basis for the developmental origins of health and disease (DOHaD) (Heindel and
474
Vandenberg, 2015).
475
In the past 15 years, mechanistic understanding of how EDCs can alter the epigenome has
476
significantly improved and three mechanisms of epigenetic modification have been identified in recent
477
EDC literature. DNA methylation and histone modification involve structural modifications to chromatin
478
that control the accessibility of genes to transcription factors. Non-coding RNAs, particularly micro-
479
RNAs (miRNAs), from the non-translated genome also regulate the epigenome and can be altered by
480
EDCs. These mechanisms are illustrated in Figure 5.
481 482
Figure 5: Mechanisms of epigenetic modifications. Chromosomes are composed of chromatin that is
483
wrapped around proteins called histone. Modifications to histone tails and direct DNA methylation can
484
control transcriptional access to DNA without modifying DNA sequence. Non-coding RNAs from the
485
untranslated genome also regulate transcription.
486
DNA Methylation
487
Modification of DNA with methyl groups is the most well-studied epigenetic modification.
488
Methylation of chromatin occurs at the 5-carbon position of cytosine to form 5-methylcytosine (5mC).
489
The cytosine is typically adjacent to guanine in the 5' → 3' direction (CpG). CpG sites are less represented
490
in the genome than expected by chance and appear in clusters known as CpG island in promoter regions
491
of genes. Methylation is generally a silencing mark, which acts by blocking access of transcription factors
492
to genes. DNA methyltransferases (DNMTs) are responsible for methylation and demethylation of CpG
493
sites. DNMT1 performs maintenance of methyl marks during replication to preserve epigenetic marks
494
through mitosis, whereas DNMT3a and DNMT3b perform de novo methylation.
495
Extensive reprogramming of methylation marks occurs during germ cell development and early
496
embryonic development (Reik et al., 2001). In both cases, methylation marks are removed and
497
remethylated, providing opportunities for disruption by environmental chemicals. Germ cell
498
modifications are heritable, which can lead to the preservation of altered methylation marks in offspring
499
and later generations, leading to transgenerational effects. For example, methoxychlor increased the
500
incidence of kidney disease, ovarian disease, and obesity as well as decreased sperm concentrations in F3
501
mice following prenatal exposure by altering methylation patterns (Manikkam et al., 2014; Stouder and
502
Paoloni-Giacobino, 2011). Gestational exposure of the F1 generation to the herbicide atrazine led to
503
increased testis disease, lean phenotype, early puberty in females, and behavioral changes in the F3
504
generation (McBirney et al., 2017). Differently methylated regions in sperm were identified for each
505
differently-exposed generation (F1 as embryo; F2 as germ cells; F3 no direct exposure), with consistently
506
differently methylated regions in the F3 generation corresponding to observation of lean phenotype
507
(McBirney et al., 2017). In sperm from F3 rats descended from F0 rats exposed gestationally to
508
vinclozolin, differentially methylated regions were associated with testis disease, prostate disease, and
509
kidney disease (Nilsson et al., 2018). In another transgenerational study of gestationally exposed rats,
510
vinclozolin induced differential methylation in sperm and brain in F1 and F3 generations (Gillette et al.,
511
2018). Adult exposure to methyl-parathion has been shown to decrease sperm quality in mice
512
immediately following exposure due to increased methylation at promoter regions of DNA repair genes as
513
well as global hypomethylation (Hernandez-Cortes et al., 2018). Although the heritability of these
514
observed changes in male germ cells was not investigated, effects due to adult exposures can be inherited
515
through the germ line. One study has examined methylation in sperm from humans exposed to TCDD,
516
finding differentially methylated regions associated with peripubertal serum TCDD measurements
517
(Pilsner et al., 2018).
518
Alternations in reprogramming during embryonic development can cause immediate toxicity or
519
increase adult diseases. The insecticide fipronil is composed of an enantiomeric mixture, one of which is
520
significantly more developmentally toxic to zebrafish. The toxic S-enantiomer hyper-methylated CpG
521
regions during development, leading to disruption of signaling pathways including MAPK, Wnt, and
522
hedgehog (Qian et al., 2017). Methoxychlor induced adult ovarian dysfunction in neonatally exposed rats.
523
Methylation analysis showed hypermethylation in ERβ promoter regions and increased Dnmt3b
524
expression (Zama and Uzumcu, 2009).
525
Histone Modification
526
DNA is organized by winding around proteins called histones to form chromatin that is compact
527
enough to fit in the nucleus of a cell (Strahl and Allis, 2000). DNA must unwind from the histones to be
528
accessible to transcription factors and thus gene expression is regulated by the structure of the histone.
529
Like methylation of DNA, histones can be modified with chemical tags that limit the ability of DNA to be
530
transcribed. Methylation and acetylation are two of the most common forms of post-transcriptional
531
modifications on histone tails that can limit the accessibility of genes.
532
Atrazine treatment of mice leads to heritable transgenerational effects on sperm mediated through
533
histone modifications. In male offspring following developmental exposure, trimethylation of the fourth
534
lysine on histone 3 (H3K4me3) was altered in promoters of key pluripotency-associated genes in sperm
535
(Hao et al., 2016). Furthermore, these marks were preserved in the F3 generation (Hao et al., 2016).
536
These marks were compared to a previous study on vinclozolin exposed mice, showing overlap in
537
differently methylated regions indicative of regions of the genome sensitive to epigenetic modifications
538
by environmental chemicals (Guerrero-Bosagna et al., 2012). Another study on atrazine exposure in adult
539
male mice found disruption of meiosis in sperm with alterations in H3K4me3 marks as well (Gely-Pernot
540
et al., 2015).
541
Recent studies have examined changes in histone modification and other epigenetic modification
542
as first steps in elucidating the mechanisms of action of less widely studied agrochemicals. The fungicide
543
carbendazim has been shown to alter spermatogenesis and steroidogenesis in mouse testes. Epigenetic
544
analysis has shown alterations in trimethylation at lysine 27 on H3 (H3K27me3) as well as changes in
545
DNA methylation (Liu et al., 2019). The organophosphate pesticide chlorpyrifos has been shown to
546
increase expression of histone deacetylase 1 (HDAC1) in mammary tissue of rats (Ventura et al., 2019).
547
The estrogenic pesticide endosulfan has also been shown to increase histone deacetylase (HDAC) and
548
DNMT expression in MCF-7 cells (K. Ghosh et al., 2018). Although each of these studies shows that
549
EDCs can act through epigenetic pathways, the detailed mechanisms through which these chemicals alter
550
expression of epigenetic regulatory proteins are largely unknown.
551
Non-Coding RNA Expression Non-coding RNAs are nucleic acids transcribed from sections of DNA that do not code for
552 553
proteins, once known as “junk” DNA. Non-coding RNAs, including small microRNAs (miRNAs), are
554
important regulators of post-transcriptional gene expression by binding to untranslated regions of mRNAs
555
to block protein translation. MiRNAs can target enzymes responsible for epigenetic marks such as
556
DNMTs and HDACs. In addition, epigenetic marks control the expression of miRNAs (Yao et al., 2019).
557
MiRNAs have been implicated in various human diseases, especially cancers (Tüfekci et al., 2014), but
558
the role of miRNAs and other non-coding RNAs is less widely studied in relation to the endocrine
559
system. However, several miRNAs that directly target ESR1 have been identified (Bhat-Nakshatri et al.,
560
2009).
561 562
Developmental exposure to atrazine in zebrafish significantly altered both human and zebrafish miRNA expression. Further analysis revealed that the altered miRNAs are associated with epigenetic
563
regulation of carcinogenesis, the cell cycle, and cell signaling (Wirbisky et al., 2016). In MCF-7 breast
564
cancer cells, DDT induced a distinct pattern of miRNA expression compared to vehicle control, estradiol,
565
or bisphenol A (BPA) (Tilghman et al., 2012). These preliminary studies show that EDCs can alter
566
miRNA expression to epigenetically regulate biological processes, but further investigations are necessary
567
for deeper understanding of the mechanism of action of EDCs on these miRNAs and identify miRNA
568
targets.
569
Conclusions
570
As this review illustrates, environmental chemicals can act as EDCs through a variety of
571
mechanisms. The diversity of pathways and precision of biological hormone actions in the endocrine
572
system makes it particularly susceptible to disruption by exogenous agents. In addition, the wide range of
573
possible phenotypes and endpoints makes integration of studies on EDCs to understand mechanisms a
574
difficult task. However, the requirement in the European Union of evidence of a plausible mode of action
575
for EDC regulation underscores the importance of mechanistic studies and analyses of these compounds
576
(Solecki et al., 2017).
577
Agrochemicals represent a significant class of EDCs that humans and animals around the world
578
are exposed to constantly. With such diversity of structures and uses, examples of agrochemicals that
579
exhibit each of the major mechanisms of endocrine disruption have been identified. Much like other
580
environmental contaminants, legacy pesticides continue to present a threat to human health, while newer
581
replacement chemicals are less well understood in terms of their endocrine disrupting potential.
582
Furthermore, newer chemicals with shorter half-lives and different routes of exposure pose challenges to
583
understanding their mechanisms of action, much like other classes of replacement EDCs.
584
Given the diversity of mechanisms through which natural hormones and EDCs act, it is vital that
585
21st century toxicology incorporates the principles of endocrinology into assessment of the safety of
586
chemicals in our environment (Schug et al., 2016; Vandenberg et al., 2019, 2013, 2012). Future studies
587
should recognize the prevalence of non-monotonic dose response curves and the importance of low dose
588
studies. In addition, mechanistic studies are needed on newer chemicals on the market and suspected
589
EDCs; legacy chemicals and controversial EDCs with lots of public interest have received most of the
590
scientific attention to date. Improvements in assays and techniques to elucidate EDC mechanisms of
591
action for computational, in vitro, and whole animal studies will facilitate interdisciplinary cooperation to
592
identify additional unstudied mechanisms of action and help regulators and the public understand the
593
various modes of action through which EDCs operate.
594
Acknowledgements
595
Thank you to Katie Chiang for Figure 5. This work was supported by Endocrine, Developmental, and
596
Reproductive Toxicology Training Grant NIH T32 ES 007326 and NIH R01 ES028661.
597
Competing interests
598
The authors declare no competing interests.
599 600
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S0303-7207(19)30382-X
DOI:
https://doi.org/10.1016/j.mce.2019.110680
Reference:
MCE 110680
To appear in:
Molecular and Cellular Endocrinology
Received Date: 1 October 2019 Revised Date:
6 December 2019
Accepted Date: 10 December 2019
Please cite this article as: Warner, G.R., Mourikes, V.E., Neff, A.M., Brehm, E., Flaws, J.A., Mechanisms of action of agrochemicals acting as endocrine disrupting chemicals, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2019.110680. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Abstract Submission MCE-D-19-00600 Mechanisms of Action of Agrochemicals Acting as Endocrine Disrupting Chemicals
Agrochemicals represent a significant class of endocrine disrupting chemicals that humans and animals around the world are exposed to constantly. Agrochemicals can act as endocrine disrupting chemicals through a variety of mechanisms.
Recent studies have shown that
several mechanisms of action involve the ability of agrochemicals to mimic the interaction of endogenous hormones with nuclear receptors such as estrogen receptors, androgen receptors, peroxisome proliferator activated receptors, the aryl hydrocarbon receptor, and thyroid hormone receptors. Further, studies indicate that agrochemicals can exert toxicity through non-nuclear receptor-mediated mechanisms of action. Such non-genomic mechanisms of action include interference with peptide, steroid, or amino acid hormone response, synthesis and degradation as well as epigenetic changes (DNA methylation and histone modifications).
This review
summarizes the major mechanisms of action by which agrochemicals target the endocrine system.
1 2 3 4 5
Review
6 7
Mechanisms of Action of Agrochemicals Acting as Endocrine Disrupting Chemicals
8
Special Issue on “Health effects of agrochemicals acting as endocrine disrupters”
9 10
Genoa R. Warner, Vasiliki E. Mourikes, Alison M. Neff, Emily Brehm, Jodi A. Flaws
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Department of Comparative Bioscience, University of Illinois at Urbana-Champaign, Urbana, IL 61802
13 14
Corresponding author
15 16 17 18 19
Jodi A Flaws [email protected] 2001 S. Lincoln Ave Urbana, IL 61802
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22 23
Introduction In Silent Spring in 1962, Rachel Carson recognized that chemicals in the environment,
24
particularly the pesticide dichlorodiphenyltrichloroethane (DDT) and other agrochemicals, were
25
impacting reproduction and development in wildlife and had the potential to harm humans (Carson,
26
1962). Although she did not know it at the time, Rachel Carson was describing the effects of endocrine
27
disrupting chemicals (EDCs), compounds that interfere with the action of hormones in the body (Gore et
28
al., 2015). Over the past half century, scientists have identified the mechanisms through which DDT
29
causes the softening of eggshells and decreases in bird populations described in Silent Spring as well as
30
numerous other mechanisms through which environmental chemicals can interfere with the complex
31
operations of the endocrine system.
32
In this review, we summarize endocrine disruptor mechanisms of action with an emphasis on
33
agrochemicals that act as EDCs. The endocrine system operates through complex and finely tuned
34
hormone signaling pathways. As such, EDCs exhibit numerous modes of action. The most well-known
35
mechanisms of EDC action involve mimicking the interaction of endogenous hormones with nuclear
36
receptors, but evidence supporting non-nuclear receptor-mediated mechanisms of action will also be
37
discussed herein.
38
The Endocrine System
39
The endocrine system controls reproduction, development, growth, metabolism, tissue and brain
40
function, and other physiological functions in the body. Endocrine glands distributed throughout the
41
body, including the brain, thyroid, mammary glands, cardiovascular system, and reproductive organs,
42
produce and release hormones. Understanding the complexities of hormone signaling provides vital
43
context to the wide range of EDC mechanisms.
44 45
The canonical pathway of hormone signaling involves the binding of a hormone to its corresponding nuclear receptor(s). Ligand binding induces structural changes in the receptor that lead to
46
dimerization, exposure of co-factor binding sites, and DNA binding. Genomic binding may occur directly
47
to response elements in the genome (Figure 1A) or through transcription factors (Figure 1B). Hormones
48
may also function through non-genomic rapid signaling via secondary messengers (Figure 1C).
49
Exogenous chemicals, including pharmaceuticals and EDCs, can mimic hormones as ligands via any of
50
these three mechanisms as well as repress transcription by recruiting co-repressors (Figure 1D) (Heldring
51
and Pike, 2007).
52 53
Figure 1: Model of the canonical hormone signaling via nuclear receptors (NR). Hormones (grey
54
triangles) act as ligands for nuclear receptors which can bind directly to DNA (A) or indirectly via
55
transcription factors (TF) (B) to regulate gene expression. Hormones may also signal through non-
56
genomic pathways by interacting with membrane nuclear receptors or cytoplasmic receptors to initiate
57
signaling cascades via secondary messengers (SM) that lead to rapid physiological effects without
58
altering gene expression (C). Hormone mimicking chemicals may operate through any of the mechanisms
59
shown in A-C as well as recruit co-repressors to block gene transcription (D). Adapted from Helding and
60
Pike, 2007.
61
Extremely low concentrations of hormones are present in the body, on the order of pg–ng/mL for
62
estradiol, testosterone, and thyroid hormone (Vandenberg et al., 2012). Furthermore, only small
63
percentages of many circulating hormones, including steroid and thyroid hormones, are bioavailable; the
64
majority are bound to carrier proteins. The endocrine system is tuned to respond to minor changes in
65
bioavailable hormone concentrations. Signaling at these extremely low concentrations is facilitated by the
66
high affinity of hormones for their receptors as well as non-linear relationships between hormone
67
concentration, receptor occupancy, and biological effect (Welshons et al., 2003). Thus, a change in
68
hormone concentrations at low doses has a stronger effect than the same magnitude of change at a higher
69
dose. For a full discussion of this, see Vandenberg et al., 2012. In the context of understanding endocrine
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disruptor mechanisms, this means that studying low doses of EDCs (below those typically used in
71
toxicological testing) is required to identify mechanisms, and that the mechanisms may change at
72
different doses. This is evident in non-monotonic dose response curves (NMDRCs) observed in
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toxicological studies of EDCs, in which the sign of the curve changes (i.e. U or inverted U) as the dose
74
changes. NMDRCs are established for hormonal pharmaceuticals such as tamoxifen yet they continue to
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be challenged in the context of EDCs (Vandenberg et al., 2012).
76
EDC Mechanisms of Action
77
Endocrine disrupting chemicals can bind to receptors to mimic endogenous hormones, but they
78
also act by altering hormone signaling in a variety of other ways. EDCs may interact with multiple
79
receptors, including non-nuclear receptors, as agonists, in which they facilitate genomic interactions, or as
80
antagonists, in which they cause a conformational change to the receptor to block action. They may also
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trigger non-genomic signaling that is independent of nuclear receptors. Importantly, EDCs can interfere
82
with endogenous hormone synthesis and degradation to alter hormone levels. Recent studies have also
83
identified how EDCs can follow an epigenetic mode of action by altering genomic methylation and
84
histone modifications.
85
Nuclear Receptors
86
Nuclear receptors are ligand dependent transcription factors that bind hormones and exert long
87
term control of their target cell phenotype. This is in contrast to membrane receptors which elicit faster,
88
short term effects on their respective cells. Nuclear receptors play a crucial physiologic role in
89
development, organ homeostasis, metabolism, immune function, and reproduction (Balaguer et al., 2019).
90
Hormones are the endogenous ligands of most of the major classes of nuclear receptors, but EDCs may
91
also bind nuclear receptors. Nuclear receptors can act as repressors or activators of gene transcription
92
depending on ligand binding status, the identity of the ligand itself, and available coregulators.
93
Nuclear receptors are composed of several conserved structural domains, including a DNA
94
binding domain and a ligand binding domain (Weikum et al., 2018). The DNA binding domain is
95
responsible for interacting with the genome and typically interacts with specific sequences called DNA
96
response elements, whereas the ligand binding domain contains the binding pocket for small molecules.
97
Unliganded nuclear receptors can be found in the nucleus or cytoplasm and may bind to target genes,
98
resulting in recruitment of corepressors. Following endogenous ligand binding, conformational changes to
99
nuclear receptors disrupt corepressors leading to dimerization and interaction with DNA and co-
100
activators. Upon binding to DNA or interacting with tethering co-factors, nuclear receptors further
101
activate co-factors to facilitate transcription.
102
Endocrine disrupting chemicals can interact with nuclear receptors directly and elicit strong
103
biological consequences (Gore et al., 2015). Agricultural chemicals, including herbicides, insecticides,
104
rodenticides, and fungicides, are prime examples of synthetic endocrine disrupting molecules exogenous
105
to normal eukaryotic biology that interact with nuclear receptors (Beischlag et al., 2008). Table 1
106
provides examples of the biological effects of select agrochemicals that act through nuclear receptors.
107
Table 1: Agrochemical effects mediated through nuclear receptors Chemical atrazine
dibutyltin (DBT)
DDT
DDE dieldrin & aldrin
fenarimol
lindane (γ-HCH)
Mode of Action
Effect
Reference
AhR mediated
Nephrotoxicity
(Zhang et al., 2019)
PPARγ activation
Androgenic antagonist: inhibits dihydrotestosterone (DHT) binding to AR
ESR1 activation leading to altered expression of Pgr, Ccnd1, Cyp19a1 AR antagonist: inhibits DHT binding to AR AR antagonist AR antagonist leading to decrease in prostate binding protein, ornithin decarboxylase, and insulinlike-growth factor 1 mRNA levels Androgenic antagonist: inhibits DHT binding to AR
linuron
AR antagonist
methoxychlor
ESR1 and ESR2 agonist
methoxychlor procymidone
ESR1 and ESR2 agonist AR antagonist
propiconazole & tebuconazole 2,3,7,8tetrachlorodibenzodioxin (TCDD)
Pregnane X receptor (PXR) activation
2,3,7,8tetrachlorodibenzodioxin (TCDD) tributyltin (TBT)
AhR activation
AhR activation during in utero exposure
AhR activation
Increased expression of adipogenic genes, promoted adipogenic differentiation, increased lipid accumulation, decreased glucose tolerance, increased circulating leptin levels in males Decreased fertility and cryptorchidism. Reduction of testicular weight, decreased in number and motility of spermatozoa in epididymis, loss of gametes in lumen of seminiferous tubules, decreased testosterone production by testes, increased luteinizing hormone and follicle stimulating hormone Hormonal carcinogenesis in uterus and ovaries Decreased fertility and cryptorchidism Reproductive performance affected
(ChamorroGarcía et al., 2018)
(Kelce et al., 1995; Sakly, 2001)
(Kalinina et al., 2017) (Kelce et al., 1995) (Danzo, 1997)
Reduced weight of ventral prostate, seminal vesicles, and bulbourethral glands
(Vinggaard et al., 2005)
Altered testis histology
(Danzo, 1997)
Disruption of reproductive tract development, reduction of accessory gland weight, reduction of epidydimal weight, increased serum estradiol and luteinizing hormone Premature nuclear expression of ER gene in neonatal uterine epithelium Inhibition of folliculogenesis and stimulation of anti-Müllerian hormone production in the ovary In males: shortened anogenital distance, hypospadias, reduced weight and altered histology of prostate, permanent nipples, ectopic undescended testes Hepatocellular hypertrophy and hepatocellular steatosis Cleft palate Downregulation of uterine and hepatic ESR1 results in repressed estradiol function UGT1 induction resulting in decreased serum T4 concentrations and compensatory excretion of thyroid
(Lambright, 2000)
(Eroschenko et al., 1996) (Uzumcu et al., 2006) (Ostby et al., 1999) (Knebel et al., 2019) (Abbott et al., 1996) (Romkes et al., 1987) (Nishimura et al., 2002)
stimulating hormone PPARγ and retinoid X receptor (RXR) activation PPARγ induction and Esr1 repression tributyltin (TBT) vinclozolin AR antagonist
In utero exposure results in increase lipid accumulation in adipose tissue, liver, and testis Impaired metabolic functions on liver and pancreas via lipid accumulation in white adipose tissue and hepatic inflammation Hypospadias, undescended testes, delayed puberty, transgenerational prostate disease
(Grün et al., 2006) (Bertuloso et al., 2015) (Monosson et al., 1999; Christiansen et al., 2008; Anway, 2015)
108 109
The structural similarities between EDCs and endogenous hormones form the basis for nuclear
110
receptor-based mechanisms of endocrine disruption. Both classes of molecules are generally hydrophobic,
111
lipid-soluble small molecules. In addition, many of the most worrisome EDCs have structural elements
112
such as hydroxyl groups in the proper position to interact with amino acid residues in the ligand binding
113
pocket in the same manner as endogenous hormones. For example, the methoxychlor metabolite 2,2-bis-
114
(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) contains both hydroxyl groups and hydrophobic
115
aromatic regions that allow it to bind to estrogen receptors, whereas the pyrethroid deltamethrin shares a
116
diphenyl ether structural moiety with thyroid hormones (Delfosse et al., 2015; Du et al., 2010) (Figure 2).
117
Once EDCs bind a receptor, they either activate the hormone receptor, amplifying physiological hormonal
118
activity, or antagonize endogenous hormone action to block activity. Due to the structural similarities
119
between EDCs and endogenous hormones as exemplified in Figure 2, EDCs are typically investigated as
120
competitive binders in the ligand binding domain of the receptor. In addition, EDCs which are not
121
structurally similar to hormones can still directly affect nuclear receptors by stimulation or inhibition of
122
receptor expression. This leads to imbalances in endocrine homeostasis through modification of hormone
123
receptor turnover/availability.
124 125
Figure 2: Structural similarities between endogenous hormones and agrochemicals. Top: Estradiol
126
and the methoxychlor metabolite HPTE share phenolic structure (blue) that facilitates the binding of
127
HPTE to human estrogen receptor 1 (ESR1) (Delfosse et al., 2015). The second hydroxyl group of HPTE
128
does not interact in the ESR1 binding pocket in the same way as the second hydroxyl on estradiol.
129
Bottom: The thyroid hormone triiodothyronine (T3) and pyrethroid deltamethrin share diphenyl ether
130
functional groups (green) that facilitate binding to thyroid receptors (Du et al., 2010).
131
In vitro assays are valuable tools for investigating the ability of EDCs to directly interact with
132
receptors and alter transcription processes. One commonly used assay is a recombinant receptor and
133
reporter gene assay in which a luciferase reporter gene is associated with a nuclear receptor response
134
element such that binding of a ligand to the receptor triggers transcription and luciferase activity (Legler
135
et al., 1999). Although these assays are specific, responsive, and quick, they do not measure binding
136
affinity nor identify which step of the nuclear receptor activation process is disrupted (Xiang et al., 2017;
137
Judson et al., 2018). High throughput screening of nuclear receptor hormone agonists and antagonists is a
138
top priority of regulatory agencies. In the US, the Tox21 program high throughput screening program
139
utilizes multiple in vitro and in silico assays to assess ligand binding, dimerization, DNA binding, RNA
140
transcription, and other steps in the activation process (Judson et al., 2018). However useful in vitro and
141
silico models are for predicting EDC interaction with nuclear receptors, these models do not integrate
142
multiple mechanisms of endocrine disruption that can occur in a whole animals. For this reason, in vivo
143
testing is also necessary to understand mechanisms that may occur simultaneously in multiple organ
144
systems to extrapolate to human and environmental health (Gore et al., 2015).
145
Estrogen Receptors (ESR)
146
Estrogen receptor alpha and estrogen receptor beta (ESR2) have unique and overlapping
147
physiological roles that are highly tissue and cell type dependent. Both receptors are expressed in the
148
brain, lungs, uterus, ovaries, breast, heart, and intestines. ESR1 is predominantly expressed in hepatocytes
149
of the liver and the hippocampus, whereas ESR2 is predominantly expressed in the prostate, vagina, and
150
cerebellum (Taylor and Al-Azzawi, 2000). ESR1 and ESR2 have quite similar binding pockets with
151
subtle differences in amino acids at the ligand binding domain that explain the ligand selectivity between
152
the two types (Kuiper et al., 1998). Both ESR1 and ESR2 regulate gene expression in response to
153
estrogen exposure via ligand dependent or ligand independent mechanisms, and each subtype can mediate
154
unique responses to ligands.
155
Inappropriate ESR signaling can lead to increased risk of hormone-dependent cancer, impaired
156
fertility, abnormal fetal growth and development, and altered metabolism in white adipose tissue (Zhao et
157
al., 2008). Many EDCs display estrogenic activity and interfere with normal estrogen signaling mediated
158
by ESR1 and ESR2. A notable characteristic of ESRs that makes them susceptible to direct interaction
159
with EDCs is their large binding pockets and broad specificity for ligands. The binding pockets are much
160
larger than the estrogen molecule itself (Pike et al., 1999; Brzozowski et al., 1997). Although ESR1 and
161
ESR2 have similar affinities for estrogen, EDCs and other exogenous ligands may display higher affinity
162
for one subtype over the other. For example, the o,p' isomer of DDT, which is the most estrogenic isomer
163
of DDT, has a higher relative binding affinity for ESR2 than ESR1 (Kuiper et al., 1998).
164
Organochlorine pesticides, including methoxychlor and DDT, are notorious estrogenic
165
agrochemicals. They bind both ESR1 and ESR2 (Kuiper et al., 1998) and elicit ER binding to DNA both
166
directly and through tethering as described above. DDT adversely affects the female reproductive tract by
167
stimulating uterine proliferation and impairing normal follicle development (Tiemann, 2008). Although
168
DDT was banned in the 1970s, it is persistent in the environment and accumulates in adipose tissue,
169
making it a relevant threat to public health today. DDT and its dechlorination metabolite
170
dichlorodiphenyldichloroethylene (DDE) have been detected in human adipose tissue around the world
171
many years after its use was terminated (Turusov et al., 2002).
172
Methoxychlor is another potent endocrine disruptor which stimulates uterotrophic activity and
173
impairs overall fertility in rat models (Cummings, 1997). Methoxychlor itself has low binding affinities
174
for ER. However, its major metabolite HPTE is a well-studied agonist for both ESR1 and ESR2 and is
175
likely the responsible agent for the endocrine disrupting effects of methoxychlor (Gaido et al., 2000).
176
HPTE acts as an agonist for ESR1 and antagonist for ESR2 (Gaido et al., 2000). ESR1 knockout mice do
177
not respond to HPTE treatment, indicating the effects of HPTE on gene regulation in the mouse uterus are
178
dependent on ESR1. HPTE may also mediate effects through non-classical ER signaling mechanisms
179
similar to estrogens. HPTE and estrogen treatment led to similar gene expression profiles in uteri from
180
mice expressing an ESR1 mutant deficient in DNA binding, which limits ESR1 mediated gene regulation
181
to the pathway in which ESR1 tethers to DNA through transcription factors like AP-1 and Sp1 (Hewitt
182
and Korach, 2011).
183
Pyrethroids also exhibit estrogenic and antiestrogenic activity, with variable results observed in
184
tests of different chemicals in vitro, suggesting that individual screening of members of a class of
185
pesticides is necessary to identify endocrine disrupting activity (Saillenfait et al., 2016). In addition to in
186
vitro screening, whole-organism bioassays have been used to detect the estrogenic activity of EDCs. In
187
transgenic medaka expressing green fluorescent protein, the herbicide linuron elicited anti-estrogenic
188
activity, whereas fenoxycarb, a less-studied insecticide, showed no effect on estrogen, androgen, or
189
thyroid signaling (Spirhanzlova et al., 2017). Recent studies have also investigated the estrogenic
190
properties of mixtures of pesticides. An in vitro analysis using multiple screening methods of individual
191
pesticides and mixtures on ESR1 and ESR2 found additive effects of the mixtures (Seeger et al., 2016),
192
suggesting the need for further experiments on mixtures, which are more representative of human
193
exposure.
194
Androgen Receptor
195
The androgen receptor (AR) is a ligand dependent nuclear transcription factor responsible for
196
male fetal development, secondary sex characteristics at puberty, and maintenance of spermatogenesis.
197
Beyond sex differentiation, the AR plays a critical role in development and maintenance of the
198
reproductive, musculoskeletal, cardiovascular, immune, neural, and haemopoietic systems (Rana et al.,
199
2014). Consequences of dysfunctional AR include infertility (Nenonen et al., 2011), delayed puberty
200
onset (Mouritsen et al., 2013), and cryptorchidism (Davis-Dao et al., 2012). AR signaling is also involved
201
in the development of tumors in the prostate, bladder, liver, kidney and lung.
202
The fungicides vinclozolin (Gray et al., 1994), procymidone (Hosokawa et al., 1993), and
203
prochloraz (Vinggaard et al., 2002) target the AR and their phenotypic consequences have been well
204
documented. Five triazole fungicides, tebuconazole, uniconazole, hexaconazole, peneconazole, and
205
bitertanol showed anti-androgenic activity toward human AR both before and after metabolism mediated
206
by human liver microsomes (Lv et al., 2017). Many insecticides and their metabolites have also been
207
shown to inhibit androgen receptor dependent transcriptional activity. These include multiple isomers of
208
DDT, DDE, and dichlorodiphenyldichloroethane (DDD) as well as methoxychlor and HPTE (Maness et
209
al., 1998). Urea based herbicides such as linuron have also been identified as endocrine disruptors via AR
210
antagonism (Lambright, 2000; Spirhanzlova et al., 2017). Agrochemicals have also been studied for their
211
effects against AR as mixtures. Various combinations of androgen antagonists have been shown to disrupt
212
male reproductive development in a cumulative and additive matter, regardless of their individual modes
213
of action, and differences in tissue selectivity (Wilson et al., 2008).
214 215
Peroxisome Proliferator Activated Receptor
The peroxisome proliferator activated receptor (PPAR) is a ligand activated nuclear receptor that
216
participates in energy combustion via stimulation of lipid catabolism and energy storage via stimulation
217
of adipogenesis (Neschen et al., 2007). Lipophilic hormones, monounsaturated fatty acids,
218
polyunsaturated fatty acids, and eicosanoids are all endogenous ligands of PPAR (Ayisi et al., 2018).
219
Three subtypes of PPAR, PPARα, PPARβ, and PPARγ exist, with differing tissue distribution, ligand
220
specificity, physiologic role, and mechanism of action. Once bound to their endogenous ligands, PPARs
221
can induce the expression of genes and enzymes involved in lipid metabolism through both genomic and
222
non-genomic mechanisms.
223
In a study of the PPARα and PPARγ activity of 200 pesticides using in vitro reporter gene assays,
224
only three pesticides, doclofop-methyl, pyrethrins, and imazalil, exhibited PPAR agonist activity, which
225
was further confirmed in vitro (Takeuchi et al., 2006). Due to its primary physiologic role as a regulator
226
of adipogenesis and lipolysis, PPARγ is a major target of agricultural chemicals that act as obesogens.
227
Tributyltin (TBT), an antifouling agent, alters PPAR-mediated differentiation of adipocytes and promotes
228
adipogenesis in liver and adipose tissue (Maradonna and Carnevali, 2018). Exposure to dibutyltin (DBT),
229
the major metabolite of TBT in the body, binds PPARγ to accelerate adipogenesis in both human and
230
mouse mesenchymal stem cells. Interestingly, human cells were shown to be significantly more
231
responsive to DBT than mouse cells (Chamorro-García et al., 2018). The fungicide triflumizole has also
232
been shown to activate PPARγ to promote adipogenesis, acting as an obesogen in vivo (Li et al., 2012).
233
Aryl Hydrocarbon Receptor (AhR) Complex
234
The aryl hydrocarbon receptor complex includes the aryl hydrocarbon receptor (AhR) and the
235
aryl hydrocarbon receptor nuclear translocator (ARNT). Although it is a basic region-helix/loop.helix
236
(bHLH) protein and not a nuclear receptor, the AhR is a ligand dependent transcription factor with similar
237
structure and function to nuclear receptors (Nebert, 2017). Following ligand binding, the AhR forms a
238
heterodimer with ARNT that binds to DNA at xenobiotic response elements (Figure 3). For many years
239
prior to the discovery of endogenous AhR ligands, the receptor’s most notable role was metabolism of
240
exogenous, synthetic chemicals. The Ahr is activated by exogenous chemicals, and relays environmental
241
signals to the cell (Elferinks et al., 1990). Thus, many mechanisms through which the AhR mediate toxic
242
effects in the body are well studied. Much of what is known today regarding mechanisms of AhR
243
mediated modulation of gene expression has been studied in the context of dioxin metabolism. Recently,
244
endogenous ligands of AhR have been identified, whereas the receptor’s physiological roles in cell
245
proliferation and differentiation, immune response, inflammation, and regulation of circadian rhythm are
246
less understood (Nebert, 2017).
247 248
Figure 3: Mechanism of AhR signaling. Synthetic chemicals such as dioxin (green triangles) activate
249
AhR by binding to the receptor and translocating to the nucleus, where the complex dimerizes with the
250
aryl hydrocarbon receptor nuclear translocator (ARNT) and controls transcription by interacting with
251
xenobiotic response elements (XRE) in DNA. Transcriptional regulation by dioxin and other EDCs leads
252
to an upregulation of xenobiotic metabolism to create toxic metabolites that impact the immune system,
253
neurological function, reproduction, and carcinogenesis.
254
2,3,7,8-Tetrachlorodibenzodioxin (TCDD, also known as dioxin) is one of the most widely
255
studied AhR ligands. The AhR has been shown to modulate the activity of other transcription factors
256
including ESRs and ARs (Ohtake et al., 2011). In the presence of estrogens and androgens, the AhR-
257
ARNT heterodimer downregulates transcriptional events that would otherwise be upregulated in the
258
presence of these hormones. Dioxin bound AhR acts as a substrate specific adaptor component that
259
targets estrogen and androgen bound receptors for degradation by the cullin 4B ubiquitin ligase complex
260
(Ohtake et al., 2007). The opposite effect on transcription has been observed when AhR is activated in the
261
absence of estrogen and androgen hormones. The AhR-ARNT complex physically associates with
262
unliganded estrogen and androgen receptors bringing transcriptional co-activators to the promotors of
263
each of these receptors (Ohtake et al., 2003). Hexachlorobenzene, a banned but persistent dioxin-like
264
fungicide, also activates AhR to trigger both genomic and non-genomic effects (van Birgelen, 1998; Miret
265
et al., 2019).
266
Reporter gene assays of 23 pesticides and insecticides in human and rat cell lines indicated AhR
267
agonistic effects by iprodione, chlorpyrifos, and prochlorax, whereas five additional pesticides exhibited
268
mixed activity in the two cell lines (Long et al., 2003). Another reporter gene assay study identified
269
eleven pesticides that agonize AhR function, while two inhibited AhR activity (Ghisari et al., 2015).
270
Pyrethroids have structural elements reminiscent of dioxins, suggesting potential AhR binding activity
271
(Brander et al., 2016); cypermethrin has been shown to antagonize TCDD-induced AhR transactivation
272
(Ghisari et al., 2015). The fungicide propiconazole also activates AhR (Knebel et al., 2018). This has
273
been confirmed in silico, in vitro, and in vivo, wherein AhR was activated in a luciferase reporter gene
274
assay in a human cell line and further increased mRNA and enzyme expression of genes controlled by
275
AhR in the livers of rats treated with propiconazole via diet (Knebel et al., 2018).
276
Thyroid Receptors
277
Thyroid receptors (TR) are another class of ligand-dependent transcription factors. Studies show
278
that four active isoforms of thyroid receptors (TRα1, TRβ1, TRβ2, and TRβ3) bind the endogenous
279
thyroid hormones triiodothyronine (T3) and thyroxine (T4) (Zoeller, 2012). TRα and TRβ isoforms are
280
differentially expressed in tissues throughout the body. Isoform distribution is particularly important
281
during fetal brain development when thyroid hormone signaling is vital for normal development. Thyroid
282
receptors are also important for physiological control of metabolism and the cardiovascular system
283
(Zoeller, 2012).
284
Extensive evidence in humans and animals indicates that pesticides including chlorpyrifos, DDT,
285
and methoxychlor can disrupt thyroid signaling and neurodevelopment (Boas et al., 2012). However,
286
most evidence on pesticides implicates thyroid hormone synthesis, bioavailability, and metabolism for
287
these effects (Ghassabian and Trasande, 2018). Pesticides generally do not have a high degree of
288
structural similarity to thyroid hormones and thus have been less investigated as TR binders compared to
289
environmental chemicals such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers
290
(PBDEs) that have been shown bind directly to TR (Ghassabian and Trasande, 2018; Zoeller, 2007).
291
However, in vitro reporter gene assays have indicated the ability of multiple pesticides to bind to TR. Ten
292
pyrethroids or pyrethroid metabolites have demonstrated antagonistic activity against TR (Du et al.,
293
2010). In an in vitro reporter assay analysis of the binding of 21 pesticides to human TR, 13 bound
294
directly to TR, with 5 exhibiting agonistic activity, including procymidone, imidacloprid, mancozeb, and
295
atrazine, whereas 11 were antagonistic and 4 exhibited both agonistic and antagonistic activity (Xiang et
296
al., 2017).
297
Non-genomic Signaling
298
While genomic mechanisms of endocrine disruption involve activation of nuclear receptors that
299
translocate to the nucleus to modulate gene expression, EDCs also exhibit non-genomic mechanisms of
300
action. These actions are characterized as rapid effects that do not directly or initially influence gene
301
expression (Figure 1C) (Lösel and Wehling, 2003). A non-genomic effect can be observed in a much
302
shorter time frame (seconds to minutes of exposure to experimental chemical) than genomic effects that
303
are constrained by transcription and translation. These effects are also typically unaffected by
304
actinomycin D or cycloheximide treatment, which inhibit transcription and translation, respectively.
305
Many non-genomic actions are initiated at the level of the cell membrane through interactions
306
with membrane-associated receptors. The fungicides prochloraz and vinclozolin have been shown to
307
directly target the membrane androgen receptor ZIP9, a member of the solute carrier protein family, to
308
block testosterone-driven zinc influx and apoptosis in PC3 prostate cancer cells (Thomas and Dong,
309
2019). Another study has demonstrated that the herbicide atrazine and pesticide trans-nonachlor target the
310
epidermal growth factor receptor (EGFR) and block receptor activation and autophosphorylation
311
(Hardesty et al., 2018). Further protein-ligand docking simulations suggest that trans-nonachlor acts as a
312
competitive antagonist to the EGFR, whereas atrazine blocks the tyrosine kinase activity of the receptor
313
(Hardesty et al., 2018). The insecticide cypermethrin can also inhibit EGFR activity and downstream
314
MAPK activation by interfering with non-classical testosterone signaling in sertoli cells, leading to
315
reduced cell viability and proliferation (Wang et al., 2019).
316
Activation of membrane receptors triggers signal transduction cascades to regulate cellular
317
responses like changes in gene expression, proliferation, and metabolism. The mitogen activated protein
318
kinase pathway (MAPK) is a kinase cascasde activated downstream of membrane receptors for growth
319
factors and cytokines that regulate cell growth, survival, and differentiation (Morrison, 2012). Several
320
studies have shown MAPK pathway activation to be critical in mediating the effects of agricultural
321
chemicals like nonylphenol, atrazine, and cypermethrin on Sertoli cell viability, testosterone production in
322
Leydig cells, and gonadotropin production in cultured gonadotropes, respectively (Choi et al., 2014;
323
Pogrmic-Majkic et al., 2016; F. Li et al., 2018). One study observed reduced membrane associated
324
connexin43 expression and impaired inter- and intracellular signaling in liver progenitor cells treated with
325
either methyoxylchlor or vinclonzin (Babica et al., 2016). This effect was dependent on p38 MAPK
326
activity as well as activation of protein kinase C.
327
The phosphoinositide-3-kinase–protein kinase B/AKT (PI3K/AKT) pathway is another target of
328
agricultural chemicals. The PI3K/AKT pathway is often triggered upstream by activation of membrane
329
receptors targeted by growth factors and cytokines, and plays a critical role in cell proliferation, survival,
330
and metabolism (Hemmings and Restuccia, 2012). Current studies have demonstrated the suppression of
331
this signaling pathway by nonylphenol and ziram in the testes of exposed rats, resulting in the generation
332
of reactive oxygen species and increased apoptosis (Huang et al., 2016; Xie et al., 2018).
333
Another common target of agricultural chemicals is the cyclic adenosine 3′,5′-monophosphate
334
(cAMP) pathway. The second messenger cAMP accumulates in the cell through activation of adenylyl
335
cyclase by membrane associated G-protein coupled receptors (Sassone-Corsi, 2012). In turn, cAMP
336
activates protein kinase A (PKA) to regulate cell metabolism, ion transport, and gene expression
337
(Sassone-Corsi, 2012). Atrazine has been shown to increase cAMP levels in Leydig cells by inhibiting the
338
cAMP specific phosphodiesterase, an enzyme that catalyzes the hydrolysis of cAMP to the inactive 5-
339
AMP (Karmaus and Zacharewski, 2015). Excessive accumulation of cAMP by atrazine increases PKA
340
activity to modulate testosterone production in Leydig cells (Samardzija et al., 2016; Pogrmic-Majkic et
341
al., 2016; Karmaus and Zacharewski, 2015). Other chemicals, like methyoxychlor and its metabolite
342
HTPE, have been shown to inhibit follicle stimulating hormone (FSH) stimulated cAMP production,
343
resulting in reduced estrogen production (Harvey et al., 2015). Another study demonstrated that exposure
344
to a mixture containing organochlorine insecticides impaired cAMP signaling, leading to decreased
345
cAMP mediated post-translational processing of the steroidogenic acute regulatory protein to its active
346
form and reduced steroidogenesis in cultured Leydig cells (Enangue Njembele et al., 2014).
347
EDCs can also influence cell function by targeting membrane associated ion-channels to mediate
348
rapid non-genomic responses. These channels open to form a pore, allowing the passive diffusion of
349
specific ions into the cell that will mediate numerous cellular functions, including the control of electrical
350
excitability and the exocytosis of secreted proteins (Jentsch et al., 2004). Insecticides cypermethrin and
351
DDE as well as glyphosate-based herbicides have been shown to target membrane bound voltage gated
352
calcium channels and/or intracellular ryanodine receptors and increase intracellular calcium
353
concentrations (Tavares et al., 2013; De Liz Oliveira Cavalli et al., 2013; Ye et al., 2017; F. Li et al.,
354
2018). Perturbation in calcium homeostasis by agricultural chemicals has been linked to several
355
physiological effects, including reduced acrosomal integrity and mobility in sperm, increased oxidative
356
damage and decreased viability in Leydig cells, and increased glucose-stimulated insulin secretion from
357
pancreatic β-cells through generation of reactive oxygen species (Tavares et al., 2013, 2015; De Liz
358
Oliveira Cavalli et al., 2013; Chen et al., 2017). Another study demonstrated that cypermethrin induced
359
calcium influx triggered rapid activation of MAPK signaling, leading to increased gonadotropin secretion
360
from cultured pituitary cells (F. Li et al., 2018). Cypermethrin has also been shown to target voltage gated
361
sodium channels in the hypothalamus to increase gonadotropin-releasing hormone pulse frequency (Ye et
362
al., 2017).
363
The non-genomic effects of endocrine disrupting chemicals are not fully understood, and even
364
less is known about those driven by agricultural EDCs. This is partly due to the duration of treatment.
365
Many studies using agrochemicals involve long term treatment periods spanning days to weeks, which
366
make it difficult to discern the rapid non-genomic actions from the slower genomic actions. Use of shorter
367
exposure periods in in vitro culture systems and the inclusion of actinomycin D and cycloheximide will
368
greatly increase our understanding of the non-genomic mechanisms of agricultural EDCs.
369
Hormone Synthesis and Degradation
370
Hormones in the body can be characterized into three main groups depending on their major
371
components: peptide/protein hormones, steroid hormones, and amino acid analogue hormones.
372
Peptide/protein hormones are the most abundant in the body and include gonadotropin-releasing
373
hormone, growth hormone, FSH, luteinizing hormone (LH), and thyroid stimulating hormone (TSH).
374
Important steroid hormones include estrogens, progesterone, and testosterone. Lastly, amino acid
375
analogues include iodothyronines such as thyroxine (T4) and 3,5,3’-triiodothyronine (T3) and amines
376
such as dopamine and serotonin. The synthesis and degradation of peptide/protein, steroid, and thyroid
377
hormones can be negatively affected by EDCs, including agrochemicals. Interfering with hormone action
378
may cause negative downstream effects on reproductive and non-reproductive health.
379
Peptide/protein hormones
380 381
Peptide and protein hormones are composed of short and long amino acid chains, respectively. Synthesis occurs in the nucleus and cytoplasm of secretory cells via gene transcription, translation into the
382
peptide chain, and finally post-translational modifications. After hormones are synthesized and packaged
383
into secretory granules, neural and hormonal signals cause their secretion into extracellular space
384
(Malandrino and Smith, 2018). Some peptide and protein hormones are secreted in pulsatile patterns that
385
may have rhythmic changes that contribute to feedback mechanisms, such as the hypothalamic-pituitary-
386
gonadotropin (HPG) axis, to control other hormone production in the body. Most peptide/protein
387
hormones are soluble in aqueous solvents and do not need carrier proteins for transport throughout the
388
blood stream. Therefore, they are susceptible to rapid protease degradation, leading to a short half-life and
389
duration of action (Malandrino and Smith, 2018). TSH, a glycoprotein hormone that stimulates the
390
production of T4, is susceptible to EDC influence. Perinatal exposure in rats to a glyphosate-based
391
herbicide resulted in decreased TSH levels, decreased gene expression of deiodinases and transporters,
392
and altered other genes regulated by thyroid hormones or involved in thyroid hormone metabolism in
393
male offspring (de Souza et al., 2017). Epidemiological studies of agricultural workers have shown
394
associations between pesticide exposure and levels of FSH and LH (Recio et al., 2005; Cremonese et al.,
395
2017). Extensive evidence in animal studies also indicates that chlorpyrifos treatment alters levels of FSH
396
and LH, in addition to steroid hormones (Li et al., 2019).
397
Steroid Hormones
398
Most steroid hormone synthesis takes place in the adrenal glands, ovary, testes, placenta, and
399
adipose tissue. The synthesis of steroid hormones is known as steroidogenesis. Cholesterol is the pre-
400
cursor for all steroid hormones, and it is transferred across the mitochondrial membrane by steroidogenic
401
acute regulatory protein (StAR). Cholesterol is then converted to pregnenolone in a process that is
402
common to all steroidogenic pathways (Miller and Auchus, 2011). After the production of pregnenolone,
403
the pathway varies depending on the hormone being produced; pregnenolone may undergo various
404
biotransformations to form aldosterone and cortisol in the adrenal gland or progesterone, estradiol, and
405
testosterone in the gonads (Acconcia and Marino, 2018; Auchus, 2014) (Figure 4). Most steroids are
406
hydrophobic in nature so hormones and their precursors can leave steroidogenic cells easily and are not
407
stored (Acconcia and Marino, 2018). After a biological response occurs, hormone secretion ceases which
408
contributes to feedback loops in the body, including the HPG axis and the hypothalamic-pituitary-adrenal
409
(HPA) axis (Acconcia and Marino, 2018). Carrier proteins transport steroid hormones throughout the
410
body and control the proportions of bound and unbound hormones (Leung and Farwell, 2018). Steroid
411
hormones are eliminated through enzymatic reactions followed by transport across the cell membrane.
412
Many of these reactions occur in the liver and include hydroxylation, conjugation, and reduction-
413
oxidation (You, 2004). After metabolites reach circulation, they are readily excreted in both urine and bile
414
as unconjugated or conjugated metabolites, but some may remain in circulation by binding to serum
415
proteins (You, 2004).
416 417
Figure 4: Steroidogenesis. Cholesterol is imported by steroidogenic acute regulatory protein (StAR) and
418
transformed into pregnenolone, which is further converted to adrenal and gonadal hormones.
419
Agrochemical exposure causes detrimental effects on the process of steroidogenesis that occurs in
420
the testis, ovary, and adrenal gland. These effects have been observed in both in vivo and in vitro
421
experimental designs. Alteration of steroidogenesis is a widely studied mode of action of EDCs; thus,
422
Table 2 provides examples of recent studies on the impacts of agrochemicals on steroid hormones.
423
Considering the literature, some common targets of agrochemical EDCs emerge. Multiple chemicals have
424
been shown to affect the synthesis of cholesterol, which is the pre-cursor for all steroid hormones. In the
425
adrenal glands, agrochemicals negatively affect the hormones, genes, and proteins involved in the
426
synthesis of steroid hormones. In the ovary, agrochemicals disrupt the production of estradiol by altering
427
the levels of enzymes and pre-cursor hormones involved in steroidogenesis in both in vivo and in vitro
428
studies. In the testes, agrochemicals impair the synthesis of testosterone, leading to detrimental effects on
429
reproduction. Table 2: Agrochemical effects on steroid hormone synthesis, metabolism, and concentrations Chemical
Mode of Action
Effect
Reference
2,4-D
Decreased serum testosterone levels, testis testosterone levels, Cyp17a1 levels, and total cholesterol levels in Leydig cells
Decreased pregnancy rate and number of pups
(Harada et al., 2016)
Decreased luminal spermatozoa
(Zhang et al., 2017)
Decreased testis, seminal vesicle, and prostate weight
(Pogrmic et al., 2009)
Altered dilation of seminiferous tubules and decreased size of Leydig cells
(Victor-Costa et al., 2010)
Altered steroidogenesis and aromatase activity
(Tinfo et al., 2011)
Decreased percent cell viability in Leydig cells
(Abarikwu et al., 2011)
Decreased levels of estradiol, but increased levels of progesterone in granulosa cells
Impaired reproductive efficiency
(Basini et al., 2012)
Altered levels of steroidogenic enzymes and increased levels of progesterone and testosterone in Leydig cells
Altered steroidogenesis
(Forgacs et al., 2013)
Increased levels of estrogen and androgens in adrenal cells
Interference with androgen synthesis
(Háhn et al., 2016)
Reduced levels of 3β-HSD expression
Decreased testis weight and heterogeneous testis morphology Decreased female fertility
(MartinsSantos et al., 2017) (PogrmicMajkic et al., 2018) (Ji et al., 2019)
Decreased serum testosterone levels atrazine
Decreased steroidogenic enzymes in Leydig cells, decreased serum levels of dihydrotestosterone and testosterone, decreased transport of cholesterol to mitochondria Decreased testosterone levels and increased estradiol levels, decreased protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) expression Increased estradiol and progesterone levels in granulosa cells, increased estradiol, estrone, and progesterone levels in adrenal cells Altered levels of steroidogenic enzymes
Reduced levels of steroidogenic enzymes
cypermethrin
Altered levels of steroidogenic enzymes, increased levels of estrogen, cortisol, and aldosterone in adrenal cells
Altered the steroidogenic pathway
Increased levels of steroidogenic enzymes, increased levels of luteinizing hormone, follicle-stimulating hormone, and testosterone
Accelerated onset of puberty in males
Altered levels of steroidogenic enzymes, decreased levels of testosterone, increased levels of estradiol in vivo and in vitro
Altered testis weight over time, decreased germ cells, impaired spermatogenesis and steroidogenesis Decreased testis weight, sperm count, sperm motility, sperm viability, sperm production, and inhibited spermatogenesis Impaired spermatogenesis and decreased sperm count
endosulfan
Decreased steroidogenic enzyme and testosterone levels
fenvalerate
Decreased expression of steroidogenic enzymes and decreased serum and testicular testosterone Decreased levels of corticosterone, testosterone, cholesterol, but increased weight of adrenal glands Altered levels of steroidogenic enzymes, decreased progesterone levels, but increased estradiol levels Increased levels of cholesterol but decreased levels of steroidogenic enzymes in the ovary, decreased levels of cholesterol, but increased levels of steroidogenic enzymes in the adrenal glands, and deceased serum levels of LH, FSH, estradiol, and progesterone Decreased levels of steroidogenic enzymes and levels of testosterone, FSH, and LH
glyphosate and glyphosatebased herbicides lambdacyhalothrin
mancozeb
Decreased levels of steroidogenic enzymes and levels of testosterone
Decreased testosterone and FSH levels
methoxychlor
ziram
Altered levels of steroidogenic enzymes, decreased testosterone levels, increased estradiol levels Decreased levels of steroidogenic enzymes and testosterone Decreased levels of most steroidogenic enzymes and decreased levels of estradiol, testosterone, androstenedione, and progesterone Decreased expression of steroidogenic enzymes, levels of testosterone, and levels of FSH Decreased steroidogenic enzyme, testosterone, and FSH levels
430 431
Amino Acid Hormones
Disrupted function of HPA axis
Decreased ovary weight, altered ovarian histology, and sex ratio of pups Altered ovarian structural degenerations and follicular maturation
Delayed Leydig cell development Decreased testis, epididymis, seminal vesicle, vas deferens, and prostate weight, decreased sperm parameters Increased abnormal sperm morphology and sperm viability Decreased testis weight and disrupted spermatogenesis
(Ye et al., 2017) (Huang and Li, 2014)
(Aly and Khafagy, 2014)
(Zhang et al., 2010) (Pandey and Rudraiah, 2015) (Ren et al., 2018) (R. Ghosh et al., 2018)
(H. Li et al., 2018) (Girish and Reddy, 2018)
(Elsharkawy et al., 2019) (Du et al., 2014)
Decreased testis weight, cauda sperm count, and sperm motility Decreased antral follicle growth
(Aly and Azhar, 2013)
Decreased epididymis and testis weight
(Guo et al., 2017)
Disrupted Leydig cell development
(Xie et al., 2018)
(Basavarajappa et al., 2012)
432
Amino acid analogue hormones develop from amino acids. Specifically, the amines are derived
433
from tyrosine and are secreted from both the thyroid and adrenal medulla (Koibuchi, 2018). The thyroid
434
gland synthesizes the endogenous thyroid hormones T4 and T3, which require dietary iodine. Thyroid
435
hormones are produced extracellularly in the follicular lumen (Koibuchi, 2018). The lumen is filled with
436
the glycoprotein thyroglobulin, which is synthesized within the epithelial cells and secreted into the
437
lumen (Koibuchi, 2018). Iodine is oxidized by the enzyme thyroperoxidase and then binds to tyrosine
438
residues in the thyroglobulin, resulting in the formation of iodotyrosines (Koibuchi, 2018; Kopp et al.,
439
2008). The iodotyrosines are coupled by thyroperoxidase to form T4, which is further converted into T3
440
in target tissues by deiodinases. Thyroid hormones are transported via carrier proteins including thyroid
441
binding globulin and transthyretin. The levels of T4 and T3 are regulated by deiodinases in peripheral
442
tissues and metabolism in the liver (Boas et al., 2012).
443
Although pesticides are generally poor structural mimics for thyroid hormones, they have been
444
shown to exert significant effects on thyroid hormone synthesis, concentrations, and carrier proteins.
445
Nonylphenol, a now-banned surfactant previously used in pesticide formulations, inhibits thioperoxides
446
necessary for the synthesis of T4. (Schmutzler et al., 2004) The triazole herbicide amitrole has been
447
shown to increase the transcription of thyroglobulin gene, while decreasing the uptake of iodide (Pan
448
Hongmei et al., 2011). Further, DDT has been shown to down-regulate the iodine-accumulation function
449
of follicular thyrocytes by suppressing sodium/iodide symporter synthesis and disrupting regular thyroid
450
function in male rats (Yaglova and Yaglov, 2015). Multiple human epidemiological studies have
451
indicated an association between disrupted thyroid hormone levels and pesticide use (Cremonese et al.,
452
2017; Recio et al., 2005; Piccoli et al., 2016; Blanco-Muñoz et al., 2016; Lacasaña et al., 2010).
453
Malathion, nonylphenol, ioxynil, and pentachlorophenol exposure decreased T3 binding to Japanese quail
454
transthyretin (TTR), one of the carrier proteins, with no observed interaction with the ligand binding
455
domain of thyroid receptors (Ishihara, Nishiyama, et al., 2003). Dicofol was shown to alter T3 binding to
456
bullfrog TTR in a biphasic manner, with low doses of dicofol increasing binding and high doses
457
inhibiting binding (Ishihara, Sawatsubashi, et al., 2003). Recently, enhanced binding of T4 to human TTR
458
has been demonstrated in the presence of organophosphate triesters, which are typically used as flame
459
retardants, but have similar structures to organophosphate pesticides (Hill et al., 2018).
460
Epigenetics
461
Epigenetics encompasses changes in gene expression without changing DNA sequences. The
462
epigenome, which consists of all chemical and structural marks that control the accessibly of the genome,
463
is highly sensitive to environmental conditions including nutrition and stress as well as chemical
464
exposures. Changes to the epigenome can be heritable and can manifest in health impacts and disease
465
long after the exposure has ended, even multiple generations after the exposure occurred (Rattan and
466
Flaws, 2019). Observed transgenerational effects of environmental chemicals were one of the first
467
indications that EDCs could alter the epigenome. For example, vinclozolin and methoxylchlor exposure
468
during pregnancy have been shown to decrease fertility in male descendants for up to four generations
469
(Anway et al., 2005). The effects of these chemicals on germ cell genomes make these outcomes
470
heritable. Development is also a particularly sensitive window of exposure due to the extensive genetic
471
programming that occurs in this time period. Exposures during periods of development can alter the
472
epigenome to increase susceptibility to disease later in life, after the exposure has ended. This forms the
473
mechanistic basis for the developmental origins of health and disease (DOHaD) (Heindel and
474
Vandenberg, 2015).
475
In the past 15 years, mechanistic understanding of how EDCs can alter the epigenome has
476
significantly improved and three mechanisms of epigenetic modification have been identified in recent
477
EDC literature. DNA methylation and histone modification involve structural modifications to chromatin
478
that control the accessibility of genes to transcription factors. Non-coding RNAs, particularly micro-
479
RNAs (miRNAs), from the non-translated genome also regulate the epigenome and can be altered by
480
EDCs. These mechanisms are illustrated in Figure 5.
481 482
Figure 5: Mechanisms of epigenetic modifications. Chromosomes are composed of chromatin that is
483
wrapped around proteins called histone. Modifications to histone tails and direct DNA methylation can
484
control transcriptional access to DNA without modifying DNA sequence. Non-coding RNAs from the
485
untranslated genome also regulate transcription.
486
DNA Methylation
487
Modification of DNA with methyl groups is the most well-studied epigenetic modification.
488
Methylation of chromatin occurs at the 5-carbon position of cytosine to form 5-methylcytosine (5mC).
489
The cytosine is typically adjacent to guanine in the 5' → 3' direction (CpG). CpG sites are less represented
490
in the genome than expected by chance and appear in clusters known as CpG island in promoter regions
491
of genes. Methylation is generally a silencing mark, which acts by blocking access of transcription factors
492
to genes. DNA methyltransferases (DNMTs) are responsible for methylation and demethylation of CpG
493
sites. DNMT1 performs maintenance of methyl marks during replication to preserve epigenetic marks
494
through mitosis, whereas DNMT3a and DNMT3b perform de novo methylation.
495
Extensive reprogramming of methylation marks occurs during germ cell development and early
496
embryonic development (Reik et al., 2001). In both cases, methylation marks are removed and
497
remethylated, providing opportunities for disruption by environmental chemicals. Germ cell
498
modifications are heritable, which can lead to the preservation of altered methylation marks in offspring
499
and later generations, leading to transgenerational effects. For example, methoxychlor increased the
500
incidence of kidney disease, ovarian disease, and obesity as well as decreased sperm concentrations in F3
501
mice following prenatal exposure by altering methylation patterns (Manikkam et al., 2014; Stouder and
502
Paoloni-Giacobino, 2011). Gestational exposure of the F1 generation to the herbicide atrazine led to
503
increased testis disease, lean phenotype, early puberty in females, and behavioral changes in the F3
504
generation (McBirney et al., 2017). Differently methylated regions in sperm were identified for each
505
differently-exposed generation (F1 as embryo; F2 as germ cells; F3 no direct exposure), with consistently
506
differently methylated regions in the F3 generation corresponding to observation of lean phenotype
507
(McBirney et al., 2017). In sperm from F3 rats descended from F0 rats exposed gestationally to
508
vinclozolin, differentially methylated regions were associated with testis disease, prostate disease, and
509
kidney disease (Nilsson et al., 2018). In another transgenerational study of gestationally exposed rats,
510
vinclozolin induced differential methylation in sperm and brain in F1 and F3 generations (Gillette et al.,
511
2018). Adult exposure to methyl-parathion has been shown to decrease sperm quality in mice
512
immediately following exposure due to increased methylation at promoter regions of DNA repair genes as
513
well as global hypomethylation (Hernandez-Cortes et al., 2018). Although the heritability of these
514
observed changes in male germ cells was not investigated, effects due to adult exposures can be inherited
515
through the germ line. One study has examined methylation in sperm from humans exposed to TCDD,
516
finding differentially methylated regions associated with peripubertal serum TCDD measurements
517
(Pilsner et al., 2018).
518
Alternations in reprogramming during embryonic development can cause immediate toxicity or
519
increase adult diseases. The insecticide fipronil is composed of an enantiomeric mixture, one of which is
520
significantly more developmentally toxic to zebrafish. The toxic S-enantiomer hyper-methylated CpG
521
regions during development, leading to disruption of signaling pathways including MAPK, Wnt, and
522
hedgehog (Qian et al., 2017). Methoxychlor induced adult ovarian dysfunction in neonatally exposed rats.
523
Methylation analysis showed hypermethylation in ERβ promoter regions and increased Dnmt3b
524
expression (Zama and Uzumcu, 2009).
525
Histone Modification
526
DNA is organized by winding around proteins called histones to form chromatin that is compact
527
enough to fit in the nucleus of a cell (Strahl and Allis, 2000). DNA must unwind from the histones to be
528
accessible to transcription factors and thus gene expression is regulated by the structure of the histone.
529
Like methylation of DNA, histones can be modified with chemical tags that limit the ability of DNA to be
530
transcribed. Methylation and acetylation are two of the most common forms of post-transcriptional
531
modifications on histone tails that can limit the accessibility of genes.
532
Atrazine treatment of mice leads to heritable transgenerational effects on sperm mediated through
533
histone modifications. In male offspring following developmental exposure, trimethylation of the fourth
534
lysine on histone 3 (H3K4me3) was altered in promoters of key pluripotency-associated genes in sperm
535
(Hao et al., 2016). Furthermore, these marks were preserved in the F3 generation (Hao et al., 2016).
536
These marks were compared to a previous study on vinclozolin exposed mice, showing overlap in
537
differently methylated regions indicative of regions of the genome sensitive to epigenetic modifications
538
by environmental chemicals (Guerrero-Bosagna et al., 2012). Another study on atrazine exposure in adult
539
male mice found disruption of meiosis in sperm with alterations in H3K4me3 marks as well (Gely-Pernot
540
et al., 2015).
541
Recent studies have examined changes in histone modification and other epigenetic modification
542
as first steps in elucidating the mechanisms of action of less widely studied agrochemicals. The fungicide
543
carbendazim has been shown to alter spermatogenesis and steroidogenesis in mouse testes. Epigenetic
544
analysis has shown alterations in trimethylation at lysine 27 on H3 (H3K27me3) as well as changes in
545
DNA methylation (Liu et al., 2019). The organophosphate pesticide chlorpyrifos has been shown to
546
increase expression of histone deacetylase 1 (HDAC1) in mammary tissue of rats (Ventura et al., 2019).
547
The estrogenic pesticide endosulfan has also been shown to increase histone deacetylase (HDAC) and
548
DNMT expression in MCF-7 cells (K. Ghosh et al., 2018). Although each of these studies shows that
549
EDCs can act through epigenetic pathways, the detailed mechanisms through which these chemicals alter
550
expression of epigenetic regulatory proteins are largely unknown.
551
Non-Coding RNA Expression Non-coding RNAs are nucleic acids transcribed from sections of DNA that do not code for
552 553
proteins, once known as “junk” DNA. Non-coding RNAs, including small microRNAs (miRNAs), are
554
important regulators of post-transcriptional gene expression by binding to untranslated regions of mRNAs
555
to block protein translation. MiRNAs can target enzymes responsible for epigenetic marks such as
556
DNMTs and HDACs. In addition, epigenetic marks control the expression of miRNAs (Yao et al., 2019).
557
MiRNAs have been implicated in various human diseases, especially cancers (Tüfekci et al., 2014), but
558
the role of miRNAs and other non-coding RNAs is less widely studied in relation to the endocrine
559
system. However, several miRNAs that directly target ESR1 have been identified (Bhat-Nakshatri et al.,
560
2009).
561 562
Developmental exposure to atrazine in zebrafish significantly altered both human and zebrafish miRNA expression. Further analysis revealed that the altered miRNAs are associated with epigenetic
563
regulation of carcinogenesis, the cell cycle, and cell signaling (Wirbisky et al., 2016). In MCF-7 breast
564
cancer cells, DDT induced a distinct pattern of miRNA expression compared to vehicle control, estradiol,
565
or bisphenol A (BPA) (Tilghman et al., 2012). These preliminary studies show that EDCs can alter
566
miRNA expression to epigenetically regulate biological processes, but further investigations are necessary
567
for deeper understanding of the mechanism of action of EDCs on these miRNAs and identify miRNA
568
targets.
569
Conclusions
570
As this review illustrates, environmental chemicals can act as EDCs through a variety of
571
mechanisms. The diversity of pathways and precision of biological hormone actions in the endocrine
572
system makes it particularly susceptible to disruption by exogenous agents. In addition, the wide range of
573
possible phenotypes and endpoints makes integration of studies on EDCs to understand mechanisms a
574
difficult task. However, the requirement in the European Union of evidence of a plausible mode of action
575
for EDC regulation underscores the importance of mechanistic studies and analyses of these compounds
576
(Solecki et al., 2017).
577
Agrochemicals represent a significant class of EDCs that humans and animals around the world
578
are exposed to constantly. With such diversity of structures and uses, examples of agrochemicals that
579
exhibit each of the major mechanisms of endocrine disruption have been identified. Much like other
580
environmental contaminants, legacy pesticides continue to present a threat to human health, while newer
581
replacement chemicals are less well understood in terms of their endocrine disrupting potential.
582
Furthermore, newer chemicals with shorter half-lives and different routes of exposure pose challenges to
583
understanding their mechanisms of action, much like other classes of replacement EDCs.
584
Given the diversity of mechanisms through which natural hormones and EDCs act, it is vital that
585
21st century toxicology incorporates the principles of endocrinology into assessment of the safety of
586
chemicals in our environment (Schug et al., 2016; Vandenberg et al., 2019, 2013, 2012). Future studies
587
should recognize the prevalence of non-monotonic dose response curves and the importance of low dose
588
studies. In addition, mechanistic studies are needed on newer chemicals on the market and suspected
589
EDCs; legacy chemicals and controversial EDCs with lots of public interest have received most of the
590
scientific attention to date. Improvements in assays and techniques to elucidate EDC mechanisms of
591
action for computational, in vitro, and whole animal studies will facilitate interdisciplinary cooperation to
592
identify additional unstudied mechanisms of action and help regulators and the public understand the
593
various modes of action through which EDCs operate.
594
Acknowledgements
595
Thank you to Katie Chiang for Figure 5. This work was supported by Endocrine, Developmental, and
596
Reproductive Toxicology Training Grant NIH T32 ES 007326 and NIH R01 ES028661.
597
Competing interests
598
The authors declare no competing interests.
599 600
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