Possible Endocrine Disruptive Effects on the Nervous and Immune Systems from Exposure to Butylated Hydroxyanisole (BHA), Tebuconazole, and Genistein in Foods Julia Dankanich Degree project in biology, Master of science (2 years), 2014 Examensarbete i biologi 45 hp till masterexamen, 2014 Biology Education Centre, Uppsala University, and The Swedish National Food Agency Supervisor: Jan Örberg, Anneli Widenfalk and Kettil Svensson External opponent: Linus Forslund
Transcript
Possible Endocrine Disruptive Effects on theNervous and Immune Systems from Exposureto Butylated Hydroxyanisole (BHA),Tebuconazole, and Genistein in Foods
Julia Dankanich
Degree project in biology, Master of science (2 years), 2014Examensarbete i biologi 45 hp till masterexamen, 2014Biology Education Centre, Uppsala University, and The Swedish National Food AgencySupervisor: Jan Örberg, Anneli Widenfalk and Kettil SvenssonExternal opponent: Linus Forslund
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Table of Contents ABSTRACT ...................................................................................................................................... 3
1. INTRODUCTION .......................................................................................................................... 6 1.1. Aim of thesis ........................................................................................................................ 6
2. BACKGROUND ............................................................................................................................ 7 2.1. PUBLIC AWARENESS AND THE ENDOCRINE DISRUPTOR HYPOTHESIS .......................................................... 7 2.2. POLICY IMPLICATIONS ...................................................................................................................... 7
2.2.1. Knowledge gaps and future needs ...................................................................................... 7 2.3 SIGNIFICANCE OF THE ENDOCRINE DISRUPTOR HYPOTHESIS ...................................................................... 8 2.4. WHY ARE CHEMICALS TO BLAME? ...................................................................................................... 8
3. NEUROENDOCRINE SYSTEM ....................................................................................................... 9 3.1. OVERVIEW ..................................................................................................................................... 9 3.2. RECEPTORS IN THE NERVOUS SYSTEM ................................................................................................ 10 3.3. NEUROENDOCRINE ANATOMY ......................................................................................................... 10 3.4. NEUROENDOCRINE AXES ................................................................................................................ 11
4. IMMUNE SYSTEM ...................................................................................................................... 16 4.1. OVERVIEW ................................................................................................................................... 16 4.2. LYMPHOCYTE MATURATION AND SELECTION ...................................................................................... 17 4.3. LYMPHOCYTE ACTIVATION .............................................................................................................. 18 4.4. IMMUNE DEVELOPMENT AND PROGRAMMING ................................................................................... 18 4.5. CYTOKINES ................................................................................................................................... 19 4.6. THE INFLAMMATORY RESPONSE ....................................................................................................... 20
5. CROSSTALK BETWEEN THE IMMUNE AND NEUROENDOCRINE SYSTEMS AND ITS IMPLICATIONS IN DEVELOPMENTAL PROGRAMMING ........................................................................................... 21
APPENDIX I ................................................................................................................................... 46
APPENDIX II .................................................................................................................................. 48
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Abstract Endocrine disrupting compounds interfere with the endocrine system in intact organisms and can lead to adverse health outcomes on reproduction, development, and other body systems. The aim of this report was to consolidate previous research findings in a literature study in order to examine the effects of exposure to endocrine disrupting compounds via food on primarily the nervous and immune systems. Three compounds with known endocrine effects were studied. These compounds included the food additive butylated hydroxyanisole (BHA), the triazole fungicide tebuconazole, and the phytoestrogen genistein. These compounds were of interest because of their prevalence in food, indicating that humans are commonly exposed. Estimates based on food consumption show that humans are unlikely to be exposed to BHA and tebuconazole at doses higher than the acceptable daily intake. However, both compounds have been shown to modulate levels of intracellular Ca2+ in vitro. Although the results from in vitro studies have unclear implication for human health, modifications of calcium can interfere with cell signaling, lymphocyte activation, and the propagation of an action potential in the nervous system. Exposure to genistein varies substantially, but exposure of western consumers is estimated to occur at levels far lower than those seen to have effects in experimental animals. Based on the findings in this thesis, all three investigated compounds have endocrine disrupting properties and have the potential to exert effects on the immune and nervous systems.
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Abbreviations ACTH adrenocorticotropin ADH anti diuretic hormone ADI acceptable daily intake APC antigen presenting cell AR androgen receptor AVP arginine vasopressin AVPV anteroventral periventricular nucleus BCR B cell receptor BHA butylated hydroxyanisole CEHOS Danish centre on endocrine disrupters CH congenital hypothyroidism CRH corticotrophic releasing hormone DDT dichloro-‐diphenyl-‐trichloroethane DES Diethylstilbestrol DRP detailed review paper ED endocrine disruption EDC endocrine disrupting compound EFSA European Food and Safety Authority ER estrogen receptor FSH follicle stimulating hormone GA gestational age GC glucocorticoid GI gastrointestinal (tract) GnRH gonadotropin-‐releasing hormone GR glucocorticoid receptor HHPS hypothalamo-‐hypophysial portal system HPA hypothalamic-‐pituitary-‐adrenal HPG hypothalamic-‐pituitary-‐gonadal HPT hypothalamic-‐pituitary-‐thyroid IPCS international programme on chemical safety LBD ligand binding domain LH luteinizing hormone MH maternal hypothyroidism MHC major histocompatibility complex MPL maximum permitted level MR mineralocorticoid receptor NOAEL/NOEL no observed (adverse) effect level NTP national toxicology program OECD organization for economic co-‐operation and development P-‐gp P-‐glycoprotein PCB polychlorinated biphenyl ppb parts-‐per-‐billion PR progesterone receptor PVN parvocellular nucleus SERM selective estrogen receptor modulator SDN-‐POA sexually dimorphic nucleus of the preoptic area TCDD 2,3,7,8-‐tetrachlorodibenzodioxin TCR T cell receptor TG thyroglobulin
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TR thyroid hormone receptor TRH thyroid releasing hormone TSH thyroid stimulating hormone WHO World Health Organization
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1. Introduction Endocrine disrupting compounds (EDCs) are compounds that interfere with the action of hormones and arrest the processes that hormones facilitate, and may thereby disrupt homeostasis, modify developmental, reproductive, neurological, behavioral and immune functions in humans and animals [1]. In 2002, a report entitled Global Assessment of the State-‐of-‐the-‐Science of Endocrine Disruptors was published and developed by the International Programme on Chemical Safety (IPCS), a joint programme of the World Health Organization (WHO), the United Nations Environment Programme (UNEP), and the International Labour Organization (ILO). The IPCS report provides a working definition of an endocrine disruptor as, "…an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations. A potential endocrine disruptor is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub) populations." [1]
The purpose of the 2002 IPCS report was to consolidate the available scientific knowledge in order to determine whether effects seen in wildlife, such as reproductive toxicity, could be attributed to chemicals in the environment [2]. From the 1990s, when the issue of EDCs first blew up, until today, much progress has been made on characterizing endocrine disruptors and testing chemicals for endocrine-‐disrupting properties. Likewise, more is known about where and how EDCs target several body systems, such as the reproductive and thyroid systems. More research is still needed, and emerging findings give rise to new questions. While there is a plethora of information available about the reproductive effects of several well-‐documented synthetic chemicals that are EDCs, the effects of natural compounds such as phytoestrogens on the endocrine system are still debated. These compounds are found in soya and other plants, mimicking estrogen and acting like the hormone in humans. In addition to reproductive effects, compounds with endocrine effects may also adversely impact other body systems, such as the nervous and immune systems. Effects on these two systems have been sparsely researched, especially in comparison to other areas. Much more research is needed into what happens when certain chemicals interfere with the proper programming of these systems. Fetal programming refers to the idea that adverse environmental conditions in utero can interrupt the processes of cell differentiation and proliferation, altering fetal growth [3]. These interruptions can influence the setup of homeostatic control mechanisms, leading to long-‐lasting health effects.
1.1. Aim of thesis The aim of this thesis is to give an overview of the issues surrounding endocrine disruptors. These issues include examining the available data to determine the specific pathways with which certain compounds or groups of compounds impact the human body. Specific pathways of interest include those encompassing the neuroendocrine and immune systems. While not meant as a comprehensive report covering all possible mechanisms of action or all known details pertaining to specific body systems, this thesis summarizes and condenses details while referencing several excellent sources that can provide readers with further information. Additionally, this report also wishes to examine the individual contribution of the antioxidant butylated hydroxyanisole (BHA), the phytoestrogen genistein, and tebuconazole, a triazole
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pesticide, on pathways susceptible to endocrine disruption that may lead to adverse effects or programming defects in the neuroendocrine and immune systems.
2. Background 2.1. Public awareness and the endocrine disruptor hypothesis In 1962, Rachel Carson's Silent Spring first alerted the public about the effects of pesticides in the environment. Carson specifically singled out the pesticide DDT (dichloro-‐diphenyl-‐trichloroethane) for its eggshell thinning and cause of declining bird populations [4]. Endocrine disruption gained momentum in the 1990s when several articles detailing the health implications of compounds like polychlorinated biphenyls (PCBs) and diethylstilbestrol (DES) were published. In her book, Our Stolen Future, Dr. Theo Colborn details the history of DES, a potent estrogenic drug given to pregnant mothers to prevent spontaneous abortion/miscarriage. Years later, it was discovered that daughters who's mothers had taken DES developed an entire slew of adverse symptoms, including reproductive organ dysfunction, reduction in fertility, immune system disorders, and early onset of a rare form of cancer, vaginal clear cell adenocarcinoma [5]. Growing public concern linked chemical exposure with birth defects, sexual abnormalities, and declining human sperm counts [6]. In the mid 90s, reports in the British Medical Journal indicated a 50% drop in male sperm count based on studies from the US, Europe, and other parts of the world [7].
2.2. Policy Implications In 1996, scientists, policymakers, NGOs, and organizations like the OECD (organization for economic co-‐operation and development), WHO, and CEFIC (European chemical industry council) assembled in a workshop in Weybridge, United Kingdom, to discuss the issue of endocrine disruptors on human and environmental health [8]. Although participants succeeded in defining an endocrine disruptor, further action didn't come until the European Parliament took up the resolution to investigate endocrine disruptors in 1998. On the heels of this resolution, the European Commission developed short-‐, medium-‐, and long-‐term goals addressing the problem of EDCs. These goals were part of the European Commission Strategy for Endocrine Disruptors, which focuses on identifying the causes and consequences of endocrine disruption (ED) and on designing appropriate policy actions on the basis of the precautionary principal [8]. Currently, the goals listed in the community strategy are still valid, and the latest update to the strategy was published in August 2011. It can be accessed via http://ec.europa.eu/environment/chemicals/endocrine/pdf/sec_2011_1001.pdf (Sec(2012)1011)
2.2.1. Knowledge gaps and future needs Despite a surge in understanding, significant knowledge gaps exist in multiple facets of ED. These gaps are especially emphasized in terms of mixtures and the effects of low-‐dose exposure. Individuals are rarely exposed to isolated chemicals; most exposure occurs in the form of mixtures, of which the effects are not necessarily the sum of the effects of individual compounds [9]. Mixture effects should be kept in mind as a testament to real-‐world exposure situations. Additional issues arise in the question of identifying EDCs. Traditional testing strategies tend to establish a linear dose-‐response curve, with more dramatic effects seen at higher doses and a NOAEL/NOEL, or the no observed (adverse) effect level at a low dose. This follows the 'dose makes the poison' mantra in toxicology, illustrating that responses at high doses can predict those at low-‐doses (due to the graphical relationship). In contrast, studies of natural hormones
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and EDCs commonly show graphical responses that are not linear, such as U-‐shaped curves, where effects are seen at the extremes, to inverted U-‐shaped, where the effects occur between a particular range of concentrations. These are called non-‐monotonic responses [10]. A 'low-‐dose effect' is defined by the National Toxicology Program (NTP) as "any biological change occurring in the range of typical human exposure or occurring at doses lower than those typically used in standard testing protocols" [10]. The debate about low-‐dose effects is whether doses in this range, or exposure so low it is in the parts-‐per-‐billion (ppb) range, matter. According to Vandenberg et al., [10] epidemiological data [11-‐16] supports associations between disease endpoints and contaminants in human tissue in the ppb range, indicating that low levels of exposure matter. This observation becomes a heated debate in the realm of policy. If EDCs exert adverse effects at environmentally relevant doses, at what point can a 'safe' level of exposure be established? This problem has not yet been resolved. In addition to the graphical relationships of exposure and effect, it is important to understand the necessity of establishing testable toxicological endpoints for further examination. The ability of a compound to interact with the endocrine system may not necessarily produce an adverse outcome, and the complexity of possible pathways of interaction makes it essential to define what an adverse outcome looks like. This is something the European Food and Safety Authority (EFSA) is currently looking into. This also makes it difficult to develop test methods and screening assays: endocrine disruption is not an endpoint, but rather a mode of action. While testing and screening methods are still under development, the OECD has integrated several Test Guidelines tailored to EDCs into a Conceptual Framework. Tests are organized into five levels of complexity, largely dealing with the ability of compounds to disrupt the estrogen, androgen, and thyroid (EAT) pathways. Additionally, a detailed review paper (DRP) supplied by the OECD details guidance on testing approaches that can be used to assess the actions and toxicity of EDCs on pathways missing from current Test Guidelines. The Conceptual Framework can be found in Appendix 1, and the detailed review paper can be accessed via http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2012)23&doclanguage=en
2.3 Significance of the endocrine disruptor hypothesis According to the WHO, the principal causes of human death around the world are due to chronic, non-‐infectious diseases. This includes, but is not limited to asthma, birth defects, neurodevelopmental disorders, cancer, diabetes, obesity, cardiovascular disease, and autoimmune disorders. Many of these are increasing [1]. Adult cancers are exceedingly common, with breast cancer, prostate, cervical cancers, colorectal, stomach, liver, oesophageal, head, neck, and bladder cancers listed as the top ten most common cancers globally. In some countries rates of thyroid disease are increasing in children. Pediatric leukemia and brain cancer incidence has also increased. Many of these diseases are common in highly industrialized countries [1]. The human genome has not changed so significantly to be responsible for the rapid increase and prevalence of non-‐infectious diseases of this nature. However, the environment continuously changes, and new chemicals are introduced on a daily basis.
2.4. Why are chemicals to blame? Although lifestyle factors such as diet and exercise play a role in disease manifestation, increasing disease rates tend to be correlated with industrialization. The nature of the illnesses listed in the previous paragraph can be associated with the abnormal functioning of the endocrine system. Some classes of industrial chemicals closely resemble endogenous hormones, propagating the belief that these chemicals mimic human hormones by binding to receptors and arresting the processes that hormones facilitate. The most widely agreed upon and well-‐studied mechanism of action for endocrine disruption is in the ability of exogenous chemicals to bind to
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and activate nuclear hormone receptors, disrupting the carefully regulated process of homeostasis [2]. The activation of intracellular receptors, such as those found in the cytoplasm and nucleus, is known as the classical genomic pathway, and involves the glucocorticoid (GR), mineralocorticoid (MR), progesterone (PR), androgen (AR), and aryl hydrocarbon (AhR) receptors. The estrogen receptor (ER) is found in the nucleus [17]. Structurally, nuclear receptors contain a DNA binding domain and a ligand-‐binding domain (LBD), along with a hinge region connecting the two domains. The compact structure of the LBD is made up of 11 helices and a 12th helix (H12) acting as a movable lid 'protecting' the entrance to the binding pocket. The receptor is kept inactive by heat shock proteins or co-‐repressors bound to the ligand-‐binding domain. When a ligand interacts with the LBD, it changes the position of H12 and exposes the docking site, enabling co-‐activator proteins to bind to helices in the region [18]. This binding facilitates dimerization of the receptor with another receptor (in the case of steroids) or with retinoid X receptor (RXR). The dimer or heterodimer can then translocate into the nucleus where it interacts with hormone response elements on the promoter regions of DNA sequences, leading to the transcription of genes [19]. Additionally, ligands can work as agonists and promote transcription, or antagonists and prevent transcription. Ligands that act as agonists move H12, enabling the co-‐activators to bind to the docking site of the LBD. Ligands that are antagonists prevent the movement of H12, thereby blocking any docking site where co-‐activators can bind. An excellent review of nuclear receptors and their modulators has been written by Burris et al [17]. Hormone receptors can also be found on the cell membrane. Cell surface receptors have a similar domain structure as nuclear receptors. The ligand recognition domain is exposed on the outer surface, and a 7-‐transmembrane domain or a single transmembrane domain spans the plasma membrane. The carboxyl terminal domain is located inside the cell. Binding of a ligand to the LBD triggers the activation of intracellular second messenger systems that mediate downstream effects and ultimately lead to biological effects, such as changes in metabolism. Typically, responses triggered by membrane receptors occur much more rapidly than those triggered by nuclear receptors, nicknaming this pathway the 'rapid action,' or non-‐genomic pathway [20]. Although many chemicals implicated as EDCs act by binding to nuclear hormone receptors, adverse effects associated from EDC exposure may not end at the target organ; unfavorable outcomes can occur in different areas of the body as a result of crosstalk between several body systems, making it difficult to predict the consequences of exposure.
3. Neuroendocrine system 3.1. Overview The cells of the nervous system include neurons and their supporting glial cells [21]. Neurons are specialized to send information rapidly and consist of a cell body and axonal protrusion. An electrochemical impulse, or action potential, is sent from its starting region at the axon hillock to its ending place at the axon terminals where neurotransmitters are released into the synaptic cleft that bridges the distance between two neurons. This action potential is dependent upon an electrochemical gradient involving the rush of ions and calcium (Ca2+) into the cell, depolarizing the membrane and leading to the ultimate release of the neurotransmitters. [21]. Glial cells include microglial and astrocytes and serve as support and immune cells in the central nervous system, or CNS, providing nutrition for neurons, as well as regulating
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inflammatory processes via cytokine release. Glial cells can detect pathogens both in the CNS and in the periphery through humoral and neuroendocrine circuits, demonstrating the degree of crosstalk that occurs between the immune and neuroendocrine systems [22].
3.2. Receptors in the nervous system There are two types of neurotransmitter receptors: ionotropic (ligand-‐gated receptors) and metabotropic (G-‐protein coupled receptors). The binding of a neurotransmitter to an ionotropic receptor induces the formation of a pore in the receptor through which ions in the synapse can enter. The types of ions that can move through the pore are dependent on the neurotransmitter-‐receptor binding. For instance, the binding of acetylcholine (Ach) to nicotinic receptors enables Na+ to enter through the pore and induce an excitatory postsynaptic potential, due to the positive charge of these ions. In contrast, binding of the neurotransmitter GABA to nicotinic receptors leads to the influx of Cl-‐. Chloride anions (Cl-‐), induce an inhibitory postsynaptic potential, thereby preventing an action potential in the postsynaptic neuron [23]. The second types of neurotransmitter receptors are metabotropic receptors, or G-‐protein coupled receptors. When a neurotransmitter binds to a metabotropic receptor, a G protein on the intracellular portion of the receptor is activated, initiating the activation of second messengers in a signal transduction cascade and leading to changes in the neuron. Often, activation of muscarinic receptors, which are metabotropic receptors, initiates a signal transduction cascade that also leads to the opening of a membrane pore and allows for the transport of ions from the outside into the inside of the cell [23].
3.3. Neuroendocrine anatomy The nervous and endocrine systems are physically connected at the hypothalamus, the brain region that acts as a major regulator of homeostasis. The hypothalamus integrates internal and external factors, such as nutrition, metabolism, temperature, photoperiod, etc. and sends hormonal messages to the periphery via the pituitary gland, located just underneath [24]. The hypothalamus contains groups of neurons organized into nuclei. One major group of neurons is found in the parvocellular nucleus (PVN); these neurons mediate crosstalk with the pituitary gland and secrete the hormones oxytocin and vasopressin from the posterior lobe of the pituitary [25]. Two hypothalamic nuclei important in the programming of sexual behavior are the anteroventral periventricular nucleus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN-‐POA). These areas contain a high volume of receptors for estrogens, androgens, and progesterone. Evidence from studies in rodents indicates that neonatal imprinting of sex-‐specific behaviors is dependent upon signals from endogenous hormones binding to their respective receptors in the AVPV and SDN-‐POA during narrow critical windows. The activation of receptors in these regions sets up the proper neural circuitry and hormonal axes that enable reproduction and sexual behavior in adulthood [26]. The hypothalamus coordinates hormone release with the pituitary gland, which is composed of the posterior and anterior lobes. The posterior lobe is effectively an extension of the hypothalamus, while the anterior lobe is connected to the hypothalamus by a region of interlacing blood vessels. This network of shared blood vessels is called the hypothalamo-‐hypophyseal portal system (HHPS). When signals from the PVN have targets in the anterior pituitary, chemical messengers released from nerve terminals are leaked into the HHPS. They can then bind to receptors on the cells of the anterior pituitary [25]. Figure 3.1 details the relationship between the hypothalamus and both lobes of the pituitary and shows which hormones are released.
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Figure 3.1. The posterior pituitary is a direct extension of the hypothalamus. Neurosecretory cells from the hypothalamic parvocellular nucleus (PVN) extend into the posterior lobe, signaling the pituitary to release the hormones oxytocin and anti-‐diuretic hormone (ADH) into blood vessels. (Arginine vasopressin (AVP) released by the posterior pituitary functions as (ADH)) The anterior pituitary is a separate gland from the hypothalamus and receives messages from the CNS via the hypothalamo-‐hypophyseal portal system (HHPS). Corticotropic cells from the anterior pituitary respond to specific signals, releasing the hormones shown in the right pane of the figure [27].
3.4. Neuroendocrine Axes
3.4.1. HPA Axis An axis generally refers to a group of glands that signal each other in sequence. Endocrine axes are tightly regulated in order to maintain homeostasis and prevent large swings in hormone levels. The hypothalamic-‐pituitary-‐adrenal (HPA) axis is primarily involved with the regulation of metabolism in vertebrates. The HPA axis also plays an important role in the immune system and has effects on growth, timing of puberty, development of reproductive organs, cardiovascular effects, ionic regulation, and memory [25]. Figure 3.2 shows a schematic drawing of the HPA axis. Table 3.1 describes the hormones involved in HPA axis signaling.
Figure 3.2. Hypothalamic-‐Pituitary-‐Adrenal axis. Corticotropin releasing hormone (CRH) released from the hypothalamus stimulates the anterior pituitary to secrete adrenocorticotropin (ACTH) into the bloodstream. ACTH acts on the adrenals, leading to the synthesis of glucocorticoids (GC).
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Table 3.1. Important hormones of the HPA, HPG, and HPT axis
HPA AXIS
Hormone Site of Production
Target Tissue Function Reference
CRH-‐corticotropin releasing hormone
Hypothalamus (PVN)
CRH-‐R in corticotropic cells
Leads to release of ACTH from anterior pituitary
[25]
AVP-‐arginine vasopressin
Hypothalamus
(PVN)
V1aR in cell membrane of
anterior pituitary
Aids CRH in release of ACTH from anterior pituitary
ACTH-‐
adrenocorticotropin
Corticotropic cells of anterior
pituitary
Melanocortin
receptors in ZF/ZR cells of adrenal
cortex
Leads to secretion of
glucocorticoids and adrenal androgens
GC-‐glucocorticoids
Adrenocortical
cells
GR1/GR2 in cytoplasm of
virtually all cells
Metabolic homeostasis, immunosuppressive,
HPG AXIS
Hormone
Site of
Production
Target Tissue
Function
Reference
GnRH-‐gonadrotropin-‐releasing hormone
Hypothalamus Anterior pituitary Leads to secretion of gonadotropins FSH and LH
[25]
FSH-‐follicle stimulating hormone
Anterior pituitary
Gonads
Females: cyclic recruitment of follicles during follicular phase
Males: controls activity of Sertoli
cells & promotes spermatogenesis
LH-‐luteinizing hormone
Anterior pituitary
Gonads
Females: ovulation & formation
of corpus luteum
Males: synthesis of androgens in Leydig cells
HPT AXIS
Hormone
Site of
Production
Target Tissue
Function
Reference
TRH-‐thyrotropin releasing hormone
Hypothalamus
(PVN)
Thyrotrophs in anterior pituitary
Leads to secretion of TSH from
anterior pituitary
[25]
TSH-‐thyroid
stimulating hormone
Thyrotrophs in anterior pituitary
Follicle cells in thyroid gland
Leads to iodide uptake in thyroid
cells; synthesis, oxidation, iodination of thyroglobin;
production of thyroid hormones
Thyroid hormones T3 and T4
Thyroid colloid cells
Multiple tissue targets: receptors found in nervous
system and nearly all cells
Neurogenesis, metabolism, growth
CRH produced by the PVN is released into the HHPS vessels, where it binds to its receptor (CRH-‐R) in the corticotropic cells of the anterior pituitary. Receptor binding initiates the formation of cyclic adenosine monophosphate (cAMP), a second messenger molecule important for signal transduction pathways. The production of cAMP activates phosphokinase A (PKA), which leads
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to an increase of available calcium (Ca2+) ions. The surge in Ca2+ releases adrenocorticotropic hormone (ACTH) from cells. ACTH travels to the adrenals, where it interacts with melanocortin receptors in the adrenal cortex. In a similar pattern as CRH, binding of ACTH to its receptor leads to the synthesis of cAMP, ultimately inducing the adrenals to secrete glucocorticoids (GC) and adrenal androgens. In humans, the main glucocorticoid is cortisol [25]. There are two types of glucocorticoid receptors: the type I mineralocorticoid receptor (MR) and the type II glucocorticoid receptor (GR). Both receptors belong to the nuclear hormone superfamily of ligand-‐activated transcription factors and induce gene transcription via the classical genomic pathway. Glucocorticoid receptors can also be found on cell or mitochondrial membranes. Activation of membrane receptors initiates the rapid response, or non-‐genomic pathway, initiating second-‐messenger signal transduction cascades [3]. The HPA axis is regulated by a system of negative feedback through which circulating GCs can bind to receptors in several places, including MR in the hippocampus, CRH neurons in the PVN of the hypothalamus, and GR in pituitary corticotropes. Binding to these receptors halts the HPA axis from releasing additional signals to continue GC secretion, thus limiting how much cortisol is in the blood [25].
3.4.1.1. HPA axis and programming In utero, fetal exposure to low-‐levels of maternal GC through the placenta facilitates the normal development of the HPA axis. During development, GCs accelerate the maturation of fetal tissues and organs, initiate the development of axon terminals, aid in the remodeling of axons and dendrites, and modulate the processes behind neural survival and apoptosis. However, too high levels of GC prove to be detrimental to the sensitive fetus, as they can have negative effects on neuronal migration, leading to the inappropriate development of the cerebral cortex [3]. Additionally, studies have shown that excessive fetal exposure to elevated levels of GCs "resets" the sensitivity of the developing HPA axis by down regulating the number of GR and MR. The down regulation of receptors impairs normal feedback regulation of the HPA axis. Often, these changes persist into adulthood, with adults who were subjected to high levels of GC in utero having higher than normal blood plasma levels of cortisol. Studies indicate that long-‐term effects of elevated levels of GC are contributors of cardiovascular and metabolic diseases [3]. To prevent excess cortisol from reaching the developing fetus, the placenta synthesizes the enzyme 11β-‐hydroxysteroid dehydrogenase 2 (11β-‐HSD2). This enzyme oxidizes 80-‐90% of maternal glucocorticoids before they can cross the placenta and enter fetal circulation. The remaining 10-‐20% of maternal GC does enter fetal circulation. As gestation progresses, the expression and activity of 11β-‐HSD2 steadily increases until about gestational age 38-‐40 weeks. At this point, the activity of the enzyme sharply declines, enabling a greater amount of maternal GC to enter fetal circulation. This speeds up the organ maturation process in time for delivery. Another protective mechanism in the placenta is the expression of P-‐glycoprotein (P-‐gp) drug transporter. While unspecific to glucocorticoids, P-‐gp regulates the kinds of endogenous compounds that can be transferred between mother and fetus [3].
3.4.2. HPG axis The hypothalamic-‐pituitary-‐gonadal axis is primarily involved in reproduction and germ cell production. During early development, the HPG axis regulates the differentiation of the sex-‐specific phenotype by inducing primary sexual characteristics and sex-‐specific behavior, which is dependent on sex-‐specific expression of endogenous hormones [25]. The AVPV and SDN-‐POA expresses ER, AR, and PR in a sexually dependent manner. Female rats have a larger AVPV in comparison to males, while males have a larger SDN-‐POA [26]. Figure 3.3 shows the flow of hormones to the various glands that make up this axis. Table 3.1 describes the hormones involved in HPG axis signaling.
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Figure 3.3. Hypothalamic-‐pituitary-‐gonadal axis. Gonadotropin-‐releasing hormone (GnRH) from the hypothalamus acts on receptors in the anterior pituitary, leading to the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH act on the gonads, facilitating the synthesis of sex steroids. Gonadrotropin-‐releasing hormone (GnRH) is released from the hypothalamus and acts on receptors in the anterior pituitary. In response to GnRH, the anterior pituitary releases two gonadotropins: follicle-‐stimulating hormone (FSH) and luteinizing hormone (LH). These hormones have their target receptors in the gonads. In females, FSH recruits follicles during the follicular phase, while a surge in LH leads to ovulation and the formation of the corpus luteum. In males, LH regulates androgen synthesis in Leydig cells, while FSH controls the activity of Sertoli cells and induces spermatogenesis in combination with androgens. The functions of FSH are generally attributed to making gametes, while LH is credited with the synthesis of the sex steroids from the precursor cholesterol. Steroid hormones act by binding to nuclear receptors, but they can also activate the non-‐genomic pathway via a G-‐protein coupled receptor. The HPG axis is regulated by a system of negative feedback, where sex steroids bind to receptors in the hypothalamus and pituitary, inhibiting the production of GnRH. Additionally, inhibin hormone produced by the gonads acts on receptors in the pituitary, suppressing FSH secretion [25, 28].
3.4.2.1. HPG axis and programming During early development, sex steroids play an active role in the differentiation of a sex-‐specific phenotype, as well as in proper sexual differentiation of the brain, which is essential in achieving reproductive competence in adulthood [26]. Development of the male duct system is dependent on the expression of the SRY gene, found on the Y chromosome, during days 41 to 44 of gestation in humans. In undifferentiated gonads, both male and female ducts exist, with the Müllerian ducts giving rise to the female duct system and the Wolffian ducts giving rise to male specific organs. SRY degenerates the Müllerian ducts and leads to the expression of androgens from fetal Leydig cells. Testosterone from Leydig cells facilitates the formation of male specific sexual organs and ducts, such as the epididymis, vas deferens, seminal vesicles, prostate, and ejaculatory ducts. In the absence of SRY, Müllerian ducts persist and Wolffian ducts regress, leading to the development of ovarian follicles and uterus and vagina [29]. Male and female differences are not confined to the duct systems-‐there are also anatomical differences in the brain of males and females. These differences include total brain volume, relative sizes of hippocampus and corpus callosum, cortical thickness and symmetry, the proportion of lipid content to gray matter, and the distribution of androgen and estrogen receptors. As already mentioned, female rats have a larger AVPV in comparison to males, while male rats have a larger SDN-‐POA [26]. The AVPV controls the preovulatory surge in levels of GnRH/LH in female rats. In rats, steroid feedback in the AVPV imprints the surge in GnRH/LH before ovulation. This imprinting must occur by postnatal day 5, or female rats are unable to exhibit a surge [26]. An increase in GnRH in the pituitary prompts the release of LH, which stimulates ovulation. If females are unable to exhibit a surge in both hormones, ovulation does not occur and the female will be infertile [30]. While the function of the AVPV is less known in
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males, male rats that are neonatally stressed have a larger AVPV and are less likely to ejaculate, suggesting that these males have been feminized and that AVPV size is inversely correlated with masculine sexual behavior. Inversely, the SDN-‐POA is approximately 5-‐fold larger in male rats. Female rats given testosterone on gestational days 18-‐20 (but not earlier), or on postnatal days 2-‐5 had larger than normal SDN-‐POA volumes. Castrating male rats during postnatal day 1 leads to a smaller SDN-‐POA volume [26]. Steroid hormones drive the organization of appropriate neural pathways specific for male and female differentiation and can exert their organizational effects by binding to their receptors in the AVPV and SDN-‐POA. The disruption of receptor binding results in abnormal organization of the brain, which can be seen in rodent models where the volumes of the AVPV or SDN-‐POA have been affected, resulting in feminization of males or masculinization of females. The absence of fetal imprinting by steroid hormones prevents the occurrence of sex-‐specific mating behaviors in adulthood [26].
3.4.3. HPT axis The hypothalamic-‐pituitary-‐thyroid axis is primarily involved with the regulation of thyroid hormones. Thyroid hormones (T3 and T4) are essential for neurodevelopment, growth, and metabolism. Figure 3.4 shows the flow of hormones to the various glands that make up this axis. Table 3.1 describes the hormones involved in HPT axis signaling.
Figure 3.4. Hypothalamic-‐pituitary-‐thyroid axis. Thyrotropin releasing hormone (TRH) from the hypothalamus stimulates the anterior pituitary to release thyroid stimulating hormone (TSH). TSH acts on thyroid receptors in thyroid follicle cells and leads to the production of thyroid hormones T3 and T4. Thyrotropin releasing hormone (TRH) produced by the PVN is released into the HHPS vessels, where it binds to its receptor TRH-‐R located in the plasma membrane of thyrotrophs in the anterior pituitary. TRH-‐R is a G-‐protein coupled receptor; the phosphorylation of TRH-‐R leads to the activation of second messenger systems and downstream kinases that result in the synthesis of thyroid stimulating hormone (TSH) from anterior pituitary thyrotrophs [25]. TSH binds to cell surface receptors of thyroid follicle cells, activating second messenger signal cascades and resulting in multiple effects such as the increased uptake of iodide into thyroid cells, synthesis and oxidation of thyroglobin (TG), iodination of tyrosyl residues on TG, and production of thyroid hormones T3 and T4 [25]. Additionally, TSH stimulates the endocytosis of T3 and T4 from thyroid colloid into central circulation. Thyroid hormones are lipophilic and can passively diffuse across cell membranes in target tissues, although specialized transporters have been identified for them as well [25]. Inside the cell, thyroid hormone binds to thyroid hormone receptor (TR), which exists in three major subtypes (TRα, TRβ1, TRβ2). Receptors tend to be tissue and temporal specific. The biologically active form of thyroid hormone is T3, and deiodinases in target tissues and cells convert T4 into T3. These enzymes are also responsible for the synthesis and breakdown of thyroid hormones [25]. T3 has near equal affinity for all three subtypes, and binds with 50 fold greater affinity for
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the TR than T4. TR is part of the nuclear family of receptors, and binding of T3 activates the genomic pathway, leading to transcription of genes involved in development, growth, and metabolism. Negative feedback in the HPT axis results from the binding of T3 to TRβ, reducing levels of TRH and TSH [25].
3.4.3.1. HPT axis and programming Thyroid hormones (TH) regulate the processes behind the growth of dendrites and axons, formation of synapses, and migration and myelination of neurons [31]. In humans, generation of neurons occurs during weeks 5-‐20 of gestation; the fetus does not begin secreting its own thyroid hormones until gestational weeks 18-‐22. This means that the TH necessary for neurogenesis comes from the mother and crosses the placenta to bind to TH receptors in different developing fetal brain regions [32]. T3 is detected in the fetal brain in the first trimester, while T4 in the brain can be detected between weeks 11 and 14. Levels of type II 5-‐monodeiodinase (the enzyme that converts T4 into T3) are detected in brain tissue up until weeks 19-‐22 of gestation. Maternal T3 peak around weeks 15-‐18, coinciding with the onset of fetal thyroid hormone production [31]. Animal models of thyroid deficiency have shown that the need for TH differs among brain regions, with basal ganglia needing TH earlier than the hippocampus. Posterior regions of the cerebral cortex bind TH earlier than anterior regions. In human development, the thyroid gland first appears during the first trimester, and it is fully functional at birth [32, 33]. Severe deficiencies of TH during human development are associated with irreversible damage to multiple organ systems [33]. For instance, maternal hypothyroidism (MH) and congenital hypothyroidism (CH) are two models of TH inadequacy during gestation and early life, and are associated with cognitive and motor defects, as well as reduced IQ scores. Because the fetus begins secreting its own TH during the second half of gestation, the effects of MH typically impact development only during the first half of gestation. CH is caused by a structural or functional abnormality in the development of the thyroid gland, which has a greater impact in the brain in the second half of gestation. Additionally, TH insufficiency is associated with compromised development of the hippocampus, the area of the brain largely responsible for memory. In children with varying degrees of hypothyroidism, both MH and CH groups exhibit memory defects with differences in the types of defects. For instance, MH leads to greater difficulty in event recall, while spatial memory tasks are affected in CH groups. Additionally, the size and structural integrity of the hippocampus is affected in children with MH and CH [33]. Screening for CH is possible and effects of hypothyroidism can be made better with thyroxine supplementation. Besides lower IQ scores and effects on memory, severe mental retardation occurs if CH is left untreated [34]. Goiter is a condition often caused by lack of iodine in the diet. The enzyme thyroid peroxidase catalyzes the iodination of thyroglobulin (TG) in thyrotrophs and the oxidative coupling of diiodothyronine, resulting in thyroid hormone formation. A lack of iodine makes it impossible to iodinate TG, thereby inhibiting the production of thyroid hormones. Lack of circulating T3 and T4 interferes with the negative feedback mechanisms of the HPT axis, and the anterior pituitary incrementally produces TSH, leading to the growth of the thyroid gland. This condition is known as goiter [35].
4. Immune System 4.1. Overview The immune system can be divided into two branches, the first consisting of non-‐specialized defenses such as physical barriers (skin and mucous membranes), mechanical barriers (cilia on epithelial cells that 'flush' bacteria away), and phagocytic immune cells that engulf foreign
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bacteria. This branch is often referred to as the innate immune system. The second branch of the immune system is generally referred to as the specialized branch of the immune system, or the adaptive immune system, and primarily consists of B and T lymphocytes. These cells distinguish between the types of bacteria and viruses that make their way into the body by responding to specific recognition patterns (antigens) on the surfaces of foreign cells. In contrast to the innate immune system, adaptive immune cells have the ability to 'remember' the distinctive shapes of bacterial antigens, ensuring that defenses can be mobilized quickly and efficiently upon second exposure to pathogens [36]. Different types of B and T lymphocytes and their functions are summarized in Table 4.1.
Table 4.1. Characteristics and functions of B and T lymphocytes [37]. (CD = 'cluster of differentiation'; describes cell surface molecules. INF= 'interferons'; refers to proteins released by host cells in response to pathogens. IL= 'interleukin'; refers to proteins that mediate communication between cells; MHC = major histocompatibility complex; APC = antigen presenting cell; Treg = regulatory T cells [36-‐40].
T Cells Types Characteristics/Description Function Effector/Helper Execute immune functions; CD4+
glycoprotein expressed on surface Activate cytotoxic T cells, B cells; bind to MHC II on APC; secrete cytokines assisting in immune response
Cytotoxic CD8+ glycoprotein expressed on surface Destroy infected cells; bind to MHC I on APCs
Memory Either CD4+ or CD8+; typically express CD45RO on surface
Long-‐lived cells; quickly become effector T cells after re-‐exposure to cognate antigen
Regulatory CD4+ Treg or adaptive Treg Maintain immune tolerance
Natural Killer T cells
Contain a T cell receptor and surface marker CD1d
Release cytokines (IL-‐2, IL-‐4) and INFγ. Recognizes bacterial glycoproteins
B Cells Types Characteristics/Description Function Effector Activated B cell; contains extensive amount
of rough ER
Production of antibodies
Plasma Type of mature effector cell Rapidly dividing, short-‐lived cells with primary purpose to secrete antigens
Memory CD27+ glycoprotein expressed on surface Long-‐lived cells; quickly become effector B cells after re-‐exposure to cognate antigen
4.2. Lymphocyte maturation and selection The main task of the immune system is to distinguish host cells from non-‐host cells. A dysfunctional immune system that cannot tell the difference manifests in autoimmune disorders. In these cases, the body wreaks havoc on its own organs or cells, resulting in massive cell death. In order to prevent self-‐reactivity, there are sophisticated measures of selection processes in the maturation of B and T cells. Both B and T cells are made in primary lymphoid organs, or the organs where lymphocytes originate. B cells are manufactured in the bone marrow, while T cells are produced in the thymus. Both cell types undergo an extensive process of selection for self-‐reactivity in their respective primary lymphoid organs. B cells that react too strongly to self-‐antigens are given the opportunity to rearrange their B cell receptors (BCR). If they continue to react to self-‐antigens, they undergo apoptosis. T cells differentiate into cells expressing specific cell surface molecules, referred to as 'CD' or 'cluster of differentiation' molecules. The expression of specific CD types gives T lymphocytes their unique identities, and T cells can differentiate into CD4+ or CD8+ cells. Only lymphocytes that pass the selection process are allowed to migrate to secondary lymphoid organs, the organs where lymphocytes reside in, such as the spleen and lymph nodes.
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Here they await stimulation by Helper T cells or antigen-‐presenting cells (APC) to mature, produce antibodies, and fulfill their functions as summarized in Table 4.1 [36, 37, 40].
4.3. Lymphocyte activation Naïve B and T cells have not yet been exposed to an antigen and must be activated by APCs. Examples of APCs include macrophages, dendritic cells, epithelial cells, fibroblasts, and B lymphocytes themselves. These cells engulf bacteria, break it down into its peptide components, and present pieces of the bacterial peptides on a protein complex called the major histocompatibility complex (MHC). There are two types of MHC: the MHA I and MHC II complexes. Virtually all nucleated cells are able to present an antigen on an MHC, but only APCs are able to activate Helper T cells by presenting antigen via the MHC II complex. Presenting an antigen on the MHC I complex is a signal that infected cells uses to mark them for destruction [36, 40]. The process of activating naïve T cells involves the release of cytokines, the chemical messengers of the immune system. Essentially, activated CD4+ cells become Helper T cells; these cells co-‐stimulate naïve B cells to produce antigen-‐specific antibodies. CD8+ T cells that interact with MHC I become Cytotoxic T cells. Cytotoxic T cells release specific proteins into the infected cell to promote apoptosis. The ability of cytotoxic T cells to destroy infected cells is known as the Th1 response, or the cell-‐mediated immune response. B cells are key players of the humoral response, or the Th2 response. The two responses overlap and work together to rid the body of bacteria or other foreign entities [36, 37].
4.4. Immune development and programming Just like the nervous system, the immune system is sensitive to external disturbances during the prenatal and early postnatal periods of development. A wide range of factors can establish the trajectory, or course, of tissue function in adulthood and risk of disease. Figure 4.2 describes the relative timeline of 'critical windows' occurring in the development of the immune system. These events include the seeding of non-‐immune tissues with the precursors of specialized immune cells (or cells that will become microglia, alveolar macrophages, Kupffer cells, skin dendritic cells, Langerhans cells, testicular macrophages, specialized gastrointestinal tract immune residents) the positive and negative selection of thymocytes, macrophage maturation near birth, and the postnatal maturation of dendritic cells to protect against hyperinflammation after birth [41].
Figure 4.2. Relative timeline of windows of immune vulnerability in humans and rodents. Days and weeks refer to gestational days and weeks, respectively [42].
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Prenatal chemical exposure can alter innate immune function; mice prenatally exposed to tetrachlorodibenzodioxin (TCDD) and presented with influenza virus after birth displayed abnormal signaling of epithelial and endothelial cells in the lung, leading to excessive neutrophil-‐driven inflammation that did not resolve appropriately. Lung damage, bronchial pneumonia, and increased mortality occurred [41]. In a different animal model, perinatal exposure of Wistar rats to bisphenol A (BPA) at the level of the NOAEL (5 mg/kg bw/day) resulted in female adults with altered architecture of tight-‐junctions in between gut epithelial cells and an impaired immune response in the colon [41, 43]. Importantly, these animal studies show that prenatal exposure to chemicals can predispose the subjects to misregulated inflammation as adults; however, the symptoms may not show up until the appropriate host challenge is administered. Thus, in TCDD treated mice, lung damage and abnormal epithelial cell signaling did not occur until the mice were given influenza. Similarly, in BPA-‐treated rats, misregulated inflammation did not occur until presented with allergens in adulthood [43].
4.5. Cytokines Cytokines are exchanged throughout the interaction of B and T cells, macrophages, and the components of the innate and adaptive immune systems. Their signaling pathways facilitate inflammatory processes and responses of immune cells to various stimuli, described in Table 4.2. Cytokine signaling is not solely confined to the immune system, as nearly all nucleated cells secrete cytokines (e.g. glial cells, liver cells) [22, 44].
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Table 4.2. Cytokines of the immune system and their effects. 'IL' refers to 'interleukin' [39, 45]. Cytokine/functional class
Cell source Cell Target Primary effects Other effects
Differentiation into a Th2 cell Growth Activation and growth; isotype switching to IgE
Th1/Th2 polarization
IL-‐5 (Th2 cytokine)
T cells B cells Eosinophil
Growth and activation
Increase antibody production
IL-‐6
T cells; Macrophages; fibroblasts
T cells; B cells; Mature B cells Liver
Costimulatory molecule Growth (in humans) Acute phase reactants
B cell activation
IL-‐8 family (chemostatic)
Macrophages; epithelial cells; platelets
Neutrophils Activation and chemotaxis
Increases cell activation
IL-‐10 (anti-‐inflammatory cytokines)
T cells (Th2) Macrophages T cells
Inhibits APC activity Inhibits cytokine production
Decreases expression of inflammatory genes; decreases cytokine-‐mediated lethality
IL-‐12 (Th1 cytokine)
Macrophages; NK cells
Naïve T cells Differentiation into Th1 cell
Increases Th1 response
IFNγ (Th1 cytokine; type II interferon)
T cells; NK cells Monocytes Endothelial cells Many tissue cells, esp macrophages
Activation Activation Increased MHC I & II
Increases Th1 response
TNFα (pro-‐inflammatory cytokines)
Macrophages; T cells
Similar to IL-‐1 Similar to IL-‐1 Increases inflammatory mediators, Increases innate immune response
4.6. The inflammatory response Inflammation is the immune system’s response to stress or infection. Injured tissues generate local pro-‐inflammatory stimuli to drive acute inflammation, such as the release of histamine and secretion of cytokines. As a result of histamine release, tight junctions in between endothelial cells of blood capillaries in damaged tissues become 'leaky,' enabling proteins, leukocytes, and other molecules from the blood or lymphatics to access the site of injury. Leukocytes are attracted to the injured tissue due to the signaling of the pro-‐inflammatory cytokines IL-‐1β and TNFα. Edema in damaged tissues occurs as a result of the arrival of fluid from the vascular compartment. Endothelial cells become activated, releasing additional cytokines to attract and
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activate other cells, such as neutrophils. Endothelial cells also express tissue factors on their surfaces, which aids in coagulation. Neutrophils and other leukocytes attach themselves to the vascular endothelium, and platelets enter the vessels. Fibrin protein is deposited to 'plug' or clot the wound, followed by the aggregation of platelets in the injured area. Macrophages and phagocytes deposited into the area engulf bacteria and release proteases and oxidants. This response is terminated once the invader is destroyed or the injury is repaired. Failure to remove the agent that initiated the response will lead to chronic inflammation, which can cause tissue damage. Upon termination of acute inflammation, leukocytes and macrophages either undergo apoptosis or drain back into the lympathics [45].
In the initial stages of inflammation, the rapid release of cytokines from cells is known as 'cytokine storm.' This response of the body serves to limit or clear infections while buying time for the more efficient adaptive immune response to be established. However, in cases of severe bacterial infection, cytokine synthesis may become deregulated and the inflammatory response goes into overdrive. This leads to a condition known as sepsis, which contributes to organ failure and can be lethal. In contrast, extreme down-‐regulation of the inflammatory response can also be lethal, as this suppresses the immune system and makes the host extremely sensitive to bacterial infections [46].
In addition to activating the adaptive immune system, pro-‐inflammatory cytokines activate the hypothalamic-‐pituitary-‐adrenal axis. Glucocorticoids suppress inflammation and protect local tissues from prolonged exposure to cytokines [47, 48]. TNFα, IL-‐1β, IL-‐6, IL-‐2, and IFN-‐γ can also act directly on the pituitary and adrenals, resulting in increases in ACTH and cortisol. In cases of chronic inflammation, abnormal HPA axis response has been implicated. Some patients with rheumatoid arthritis have inappropriately low amounts of ACTH in serum in comparison to the degree of inflammation. This impairment in the production of ACTH results in decreases in cortisol and in adrenal androgens, and thus the inflammatory response is unchecked [48]. The ‘checking’ of inflammatory processes by glucocorticoids is one example of crosstalk between the immune and neuroendocrine systems. Inflammatory events occurring during critical windows of neural development are also implicated in behavioral and psychological disorders, such as autism or schizophrenia [49]. These are discussed in section 5.
5. Crosstalk between the immune and neuroendocrine systems and its implications in developmental programming Based on the development of the three major neuroendocrine axes, HPA, HPG, and HPT, it is clear that aberration in the normal patterns of development—e.g. a lack of steroid hormones binding to their appropriate receptors in the AVPV and SDN-‐POA, maternal iodine deficiency or absence of TH crossing the placenta during the first half of gestation, or too high levels of maternal GCs crossing the placenta—can lead to abnormal patterns in behavior and impaired cognitive function in the newborn, with some of these behaviors persisting to adulthood. There is also evidence that early immune insults, such as bacterial or viral infections, have permanent programming effects on the nervous system and may even be linked to psychiatric disorders [49]. Crosstalk between the immune and nervous system occurs in the form of transport of cytokines through the blood brain barrier (BBB) from the periphery and vice versa, and via the neuronal activation of the HPA axis to execute tasks in the periphery, such as the stress response (discussed in section 4.5). The CNS has its own resident immune cells in the form of microglia and astrocytes, as well as perivascular macrophages, endothelial cells, oligodendrocytes, and neurons. These cell types fulfill immune functions in the brain [49]. In an excellent review article written by Bilbo and Schwarz [49], the authors stress that cytokines are essential for
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normal synaptic plasticity and learning and memory behaviors. They emphasize the importance of IL-‐1β in the hippocampus; mice lacking IL-‐1β or its receptor have dramatically impaired hippocampal-‐dependent learning and memory. The opposite is also true. Inflated levels of IL-‐1β within the brain are associated with memory impairments, which can be seen in patients that have cognitive deficiencies and suffer from chronic inflammatory diseases like Alzheimer's, autoimmune diseases, or AIDS-‐related dementia [49]. Adult rats neonatally infected with E. coli showed no differences in the expression of IL-‐1β in comparison to non-‐neonatally infected controls. However, adult challenge with LPS (which mimics the effects of E. coli) in the neonatally infected rats immediately unregulated gene expression of IL-‐1β, its receptor, and caspase 1 (enzyme that cleaves IL-‐1β into its active form). This does not happen in control rats. As IL-‐1 is a pro-‐inflammatory cytokine (Table 4.2), researchers have suggested that early-‐life infection programs the brain to shift to a pro-‐inflammatory phenotype when presented with immune challenge in adulthood, which leads to cognitive defects and memory impairments [49]. This is consistent with the "two hit hypothesis" of schizophrenia, which postulates that the combination of underlying vulnerability and a precipitating event is needed for the illness to manifest. For instance, immune activation indirectly increases the risks of cognitive defects via the long-‐term programming of neuroimmune responses that interfere with learning and memory processes. To lend support for this hypothesis, treatment of newborn rats or mice with Poly IC (polyinosinic:polycytidylic acid: synthesis double-‐stranded RNA molecule used as a viral mimic) is used as an animal model for schizophrenia. These animals display the symptoms associated with humans who have the disorder, such as the inability to ignore irrelevant environmental stimuli and defects in reversal learning of a previously learned task. The behavioral defects in Poly IC treated animals can be reversed by acute administration of antipsychotic and psychomimetic drugs, consistent with human treatments for the disorder. Prenatal exposure to Poly IC causes changes in neurotransmitter function, such as reduced levels of GABA (an inhibitory neurotransmitter-‐section 3.2). During an object recognition task, the pattern of neuronal activation in the hippocampus of prenatally treated rats was different in comparison to controls, indicating that in addition to neurotransmission being different, the overall function of the neuronal circuit is significantly altered after prenatal treatment with Poly IC. Treatment of pregnant dams with Poly IC also exhibited effects in offspring, as the excitatory postsynaptic potential in the hippocampus was affected in pups, and offspring displayed impaired long-‐term potentiation in the hippocampus (process of synaptic strengthening) and decreases in pre-‐synaptic proteins, which could lead to disparities in synapse number [49]. The present thesis cannot describe the plethora of advancements that have been made in regard to early-‐life immune challenge and its effects on neurological pathways and behavior in adults. However, evidence from scientific literature supports the idea that exposure to compounds or agents affecting hormone levels, cytokines, and/or receptors in the developing brain leads to permanent differences in the neuronal circuitry of adults in comparison to offspring where these insults did not happen [49]. There are a number of protective mechanisms in the brain that strive to normalize development and prevent death and serious injury to the developing fetus when it is presented with sub-‐optimal conditions, which will also not be covered in this thesis. Despite protective mechanisms, development is nevertheless affected in one way or another. The goal of this report was to determine possible pathways with which chemicals can disrupt the normal developmental processes or the activities of the immune and nervous systems. These possible pathways include disturbances in programming of the HPA, HPG, and HPT axes, and disturbances in the timing and programing of immune system development. As evidence of the crosstalk between the immune and nervous systems show, disturbing organizational events in one system is enough to exert permanent changes in the other system, causing behavioral, cognitive, or reproductive disturbances.
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The second half of this thesis examines the contributions of the antioxidant butylated hydroxyanisole (BHA), the phytoestrogen genistein, and the triazole fungicide tebuconazole on adverse effects in immune and nervous system functions.
6. BHA The first compound examined is the food additive tert-‐butylhydroxyanisole (BHA). BHA is a mixture of two isomers, 2-‐tert-‐butyl1-‐4-‐hydroxyanisole and 3-‐tert-‐butyl1-‐4-‐hydroxyanisole. Their structures are shown in Figure 6.1. It is primarily used as an antioxidant and preservative in food (especially in food with a high fat content), food packaging, and some medicines [50].
Studies of the absorption, distribution, metabolism, and excretion in rats, rabbits, dogs, monkey, and humans have shown that BHA is rapidly absorbed from the gastrointestinal (GI) tract, metabolized, and excreted mainly in urine and feces. Major metabolites include glucuronides, sulphates and free phenols. tert-‐Butylhydroquinone (TBHQ) is the most relevant metabolite and is also authorized for use as an antioxidant in the EU, alone or in combination with other antioxidants, such as BHA [51]. The latest evaluation of BHA as a food additive was done by EFSA in 2011. EFSA set the acceptable daily intake (ADI) to 1.0 mg/kg bw/day, revising it from the earlier ADI of 0.5 mg/kg bw/day. The ADI of 0.5 mg/kg bw/day was based on proliferative changes in the rat forestomach, and the update was made on the premise that humans lack a forestomach. To obtain the updated ADI, EFSA used the NOAEL of 100 mg/kg bw/day for growth retardation, increased mortality, and behavioral effects observed in rat pups and an uncertainty factor of 100 [51].
6.1. Exposure analysis
6.1.1. Exposure via food Based on country-‐specific data from national dietary surveys found in the Comprehensive Food Consumption Database, EFSA was able to carry out a refined exposure assessment for BHA. 17 different European countries provided data on food consumption. Because the actual levels of BHA in food are unknown, EFSA used the maximum permitted levels (MPLs) instead to calculate exposure; using MPLs makes EFSA's exposure assessment conservative, since actual levels found in food are likely to be below the MPLs. The results of the estimated ranges of exposure based on EFSA's calculations are shown in Table 6.1.
Figure 6.1. Structure of 2-‐tert-‐butyl1-‐4-‐hydroxyanisole and 3-‐tert-‐butyl1-‐4-‐hydroxyanisole (left) compared to 17-‐β-‐estradiol (right).
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Table 6.1. Summary of estimated ranges of exposure to BHA (mg/kg bw/day) in five population groups in Europe. The values in black are an average from the data submitted by the 17 European countries. The values in red are country-‐specific results for Sweden [51]. Estimated exposure using MPLs
Toddlers
Children
Adolescents
Adults
Elderly
Mean
0.04-‐0.23 0.08-‐0.36 0.25
0.06-‐0.18 0.13
0.03-‐0.12 0.11
0.02-‐0.11
High level (95th per.)
0.14-‐0.57 0.26-‐0.60 0.47
0.12-‐0.38 0.29
0.08-‐1.12 0.21
0.05-‐0.72
6.1.2. Exposure via food contact materials BHA exposure from its use in food contact materials would be 0.43, 0.6, 1.3 and 2.5 mg/kg bw/day for adults and the elderly, adolescents, children and toddlers, respectively, assuming that consumers from all the population groups also consume 1 kg of food packed in plastics containing BHA at the maximum permitted quantity of 30 mg/kg foods [51].
6.2. BHA as an endocrine disruptor The Danish Centre on Endocrine Disruptors (CEHOS) has determined BHA to be a category 1 endocrine disruptor based on adverse effects in vivo where an endocrine disrupting mode of action was highly plausible. These effects included altered oestrous cycles and sperm morphology, and decreased sperm number in developmental in vivo rat studies [50]. In vitro, BHA reduced the binding of 17β-‐estradiol to the fish ER at concentrations up to 1 mM [52]. BHA was found to stimulate gene transcription at concentrations between 10-‐5 and 10-‐4 M [52]. In the E-‐SCREEN assay, BHA was shown to promote the proliferation of MCF7 human breast cancer cells at a concentration of 50 μM [53]. Soto et al., [53] calculated a relative proliferative effect of BHA in comparison to estradiol and found that the relative proliferative effect of BHA was 30%, indicating it is a partial agonist [53]. In the uterotrophic assay, BHA increased uteri organ weights of immature female rats at doses ranging from 50 to 500 mg/kg bw/day. However, there were no effects on androgen-‐dependent accessory sex organs of castrated male rats dosed with BHA from 50 to 500 mg/kg bw/day in the Hershberger assay [54]. An in vivo study where pregnant pigs were exposed to 0, 50, 200, or 400 mg/kg bw/day found no adverse reproductive effects on offspring at any of the doses tested. However, in the dams, thyroid and liver weights were slightly increased in the highest dose groups, and histopathological changes in the thyroid were observed at the low dose of 50 mg/kg bw/day [55]. A one-‐generational study by Jeong et al., [56] where 7-‐week old male and female Sprague-‐Dawley rats were fed by gavage BHA dissolved in corn oil at doses of 0, 10, 100, or 500 mg/kg bw/day from pre-‐breeding until weaning found that F0 males had decreased levels of serum testosterone and thyroid hormones at the 100 and 500 dose levels. F1 male and female offspring receiving exposure from the mother (at the 500 level) experienced a decrease in thyroid hormones and testosterone, along with decreased reproductive organ weights, delayed puberty and sexual maturation, decreased body and brain weights, shortened anogenital distance at post natal day 21, and dysfunction of androgen-‐dependent organs in males. Females in the F1 generation had decreased weights of reproductive organs and abnormal thyroid histology [56].
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6.3. BHA exposure and immune effects There is evidence that BHA can influence intracellular concentrations of calcium (Ca2+) [57]. Ca2+ is essential for a multitude of cellular processes, including protein kinase signaling, mitochondrial physiology, apoptosis, and cell adhesion and migration of B cells. In B lymphocytes, appropriate levels of Ca2+ lead to the translocation of transcription factor NF-‐κB into the nucleus. NF-‐κB is generally involved in the transcription of IFNγ, which increases the Th1 immune response [58]. In T cells, antigen activation is reliant on two signals: the increase in intracellular Ca2+ and the activation of protein kinase C (PKC). Studies of BHA on intracellular Ca2+ levels often produce confusing results. David et al., [59] concluded that BHA exposure in vitro at concentrations between 250 μM to 2 mM increases levels of intracellular calcium in a dose-‐dependent manner in various cell types, including human umbilical vein endothelial cells, rat cardiomyocytes, baby hamster kidney cells, and rat pituitary cells [59]. In a different in vitro study, Dornand and Gerber [57] found that BHA at a concentration of 40 μM prevented an increase in intracellular Ca2+ levels in thymocytes and splenocytes, and may affect PKC activation of T cells [57]. In Dornand's and Gerber's study [57], BHA suppressed the activation of murine thymocytes and splenocytes in a dose-‐dependent manner [57]. In T cells, sustained increases in Ca2+ allow the translocation of transcription factor NFAT from the cytoplasm and into the nucleus, where it can activate the transcription of IL-‐2 and IL-‐4 and promote the Th2 response [60]. In their study, Dornand and Gerber found that treatment of thymocytes at concentrations of 15±2 μM inhibited 50% of IL-‐2 secretion in activated T cells. 40 μM BHA decreased the expression of IL-‐2 receptor on activated splenocytes [57]. In an in vivo experiment, Hung et al.,[61] orally exposed mice to BHA. Exposed mice had increased levels of T cells, which is the opposite of Dornand and Gerber's results. In Hung's study, BHA promoted phagocytosis by macrophages in peripheral blood, but it decreased B cell levels. Due to the increase in levels of T cells and promotion of phagocytosis in normal mice, this group concluded that BHA supports immune responses. However, they stressed a need for further investigation [61].*
6.4. BHA exposure and neuroendocrine effects Vorhees et al., [62] exposed Sprague-‐Dawley rats to BHA daily in the diet from pre-‐mating until after weaning, with the F1 generation being subsequently exposed in food after weaning until 90 days of age. Doses of BHA were measured as percentages of the wet weight of the diet, and included 0.50% BHA (BHA-‐50), 0.25% (BHA-‐25), 0.125% (BHA-‐12) and control group. The BHA-‐50 group corresponds to approximately 0.42 g/kg bw/day during prebreeding and gestation to 0.80 g/kg bw/day during lactation. The BHA-‐25 group corresponds to about 0.21 g/kg bw/day from prebreeding and gestation to 0.44 g/kg bw/day during lactation. The BHA-‐12 group corresponds to about 0.10 g/kg bw/day during prebreeding and gestation to 0.22 g/kg bw/day during lactation. Exposed and control rats underwent a series of behavioral tests to determine whether BHA affected motor functions or learning, and brains of exposed rats were autopsied to determine whether there were differences in neuronal cell density in various brain regions. The autopsy results show no differences in the brains of exposed and control rats, and there were no statistically significant differences in the results of behavioral tests between BHA-‐exposed and control rats except for the auditory startle parameter. Auditory startle typically measures sensory defects, and in this case it was performed in pre-‐weaned rats [62]. In comparison to the other groups, pre-‐weaned rats in the BHA-‐50 and BHA-‐25 groups both showed delayed development of the auditory startle response by roughly a day and a half. Regardless of this delay, the researchers concluded that BHA is not a potent behavioral toxin [62].
*the original article could not be located, so these results were taken from the abstract, and thus the dose levels mice were exposed to are unknown.
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Very few studies look into the behavioral effects of BHA or the affects this compound might have on the nervous system and on the developing brain.
6.5. Conclusion According to EFSA's estimations of human exposure to BHA through food, it is unlikely that humans would be exposed to BHA at levels higher than the ADI of 1.0 mg/kg bw/day. Of course, EFSA based its exposure estimations on MPL levels, rather than on actual levels found in food because information about levels in food are lacking [51]. However, it is unlikely that levels in food would be higher than the MPLs, suggesting that the estimated exposure using MPLs offers a conservative estimation of the exposure situation to the five different population groups of toddlers, children, adolescents, adults, and elderly. Based on BHA exposure via food contact materials such as plastic lunch boxes, EFSA estimated that exposure to BHA may actually occur at levels higher than the ADI and can be up to 2.5 mg/kg bw/day [51]. According to CEHOS, BHA is a category 1 endocrine disruptor [50]. Evidence of its endocrine disrupting properties is based on in vitro assays including the E-‐SCREEN assay, where BHA led to the proliferation of human breast cancer cells [53], and in vivo studies, where BHA increased uteri weights in immature female rats [54]. BHA was also found to have effects on levels of thyroid hormones and calcium concentrations [56, 57]. Its effects on intracellular Ca2+ are difficult to categorize, as studies have led to different conclusions. However, BHA may very likely have effects on the immune system in terms of lymphocyte activation and cytokine production due to its ability to modulate calcium levels [57, 59]. There is a lack of behavioral studies in animals treated with BHA, and the few studies that look into neurobehavioral parameters of BHA exposure have found that young rats treated with BHA have delayed development of the auditory startle response [62]. Undoubtedly, there is in general a lack of studies examining the disruptive potential of BHA on the endocrine system. The in vivo studies examined in this report that showed adverse effects in exposed animals occur at exposure that is higher than the NOAEL of 100 mg/kg bw/day. Thus, it is not considered likely that the current exposure levels of consumers to BHA causes endocrine disruption.
7. Genistein The second compound examined is the phytoestrogen genistein. Phytoestrogens are non-‐steroidal compounds structurally similar to natural estrogens found in mammals. Isoflavones are the most common types of phytoestrogen and are found in soya products. Other types of phytoestrogens include coumestans, lignans, and stilbens [63]. Genistein is a specific type of isoflavone found in soya products. Soy is made infamous due to claims that it has protective effects against various cancers, such as breast and prostate cancer. However, epidemiological and animal studies have shown variable effects [64]. The structure of genistein is shown in figure 7.1.
Figure 7.1. Structure of genistein and estradiol.
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Genistein is metabolized differently in rodents, pigs, monkeys, and humans [51]. Likewise, there is large variation in individual metabolism of genistein between members of the same species [53]. In humans, genistein is rapidly absorbed following oral intake. In plants, phytoestrogens bound to a sugar derivative are in their inactive glycoside form. Upon ingestion, gut microflora convert the glycoside into the biologically active aglycone form. The activity of the aglycone is mainly confined to the GI tract, as aglycones passing through intestinal epithelial cells are conjugated with glucuronic acid or sulfate before they enter circulation. Once conjugated, the compound is no longer bioactive [65]. Roughly 10% of ingested genistein circulates in the aglycone form. Genistein aglycone can enter tissues more readily than genistein glycoside, although tissues hosting de-‐conjugation enzymes may metabolize genistein into the active form [66]. Conjugated isoflavones, genistein included, can undergo enterophepatic circulation and return to the intestine to be de-‐conjugated and metabolized further [65, 66]. There is considerable individual variation in the absorption and metabolism of genistein. The National Toxicology Program distinguishes the half-‐life between free and total genistein, with total genistein being the amount of genistein bound to glucuronic acid or sulfate. The absorption half-‐life for free genistein is estimated to be between 2 and 7 hours and between 6 and 13 hours for total genistein [66]. Excretion of isoflavones occurs mostly via urine. The excretion half-‐life for genistein is between 3 and 8 hours [67]. In infants, urinary excretion of genistein is typically lower compared to adults fed the equivalent amounts of isoflavones, indicating slower renal excretion in young individuals. After ingestion of soymilk, excretion peaked between 8 and 10 hours after consumption in infants, and 95% of the compound was recovered after 24 hours in urine [66].
7.1. Exposure Analysis In soybeans the isoflavones content varies between 560 and 3810 mg/kg depending on the variety of bean and its growing conditions, and genistein makes up approximately 50% of this content [65]. There is no ADI for soy isoflavones or phytoestrogens, and rates of consumption vary widely. Estimates for the average daily intake of soy and isoflavones in Asian countries ranges from 1 mg/kg bw/day [67] to between 20 and 150 mg/kg bw/day [64]. Japanese men and women consuming a traditional soy-‐based diet have mean plasma isoflavone concentrations of 1 μM [67]. In western countries, the daily intake of soy is estimated to be only about 2 mg to 1 g daily [64], i.e. 0.033-‐16.7 mg/kg bw for a woman with a body weight of 60 kg. Plasma concentrations of isoflavones in Europeans and North Americans are less than 0.07 μM for omnivores and 0.4 μM for vegetarians [67]. A study of Japanese infants conducted by Adlercreutz et al., [68] demonstrates that isoflavones cross the placenta and gain access to the developing fetus. In this study, concentrations of isoflavones in cord blood and in amniotic fluid were 0.2-‐0.3 μM, which was similar to maternal plasma concentrations (0.2 μM) [68]. Trace amount of isoflavones can also be found in breast milk. Concentrations of isoflavones in the breast milk of mothers eating a soy-‐rich diet range from 10 to 70 nM, while mothers who do not actively consume soy have breast milk concentrations between 18 and 56 nM [67]. The high human consumption of isoflavones occurs in infants fed soy-‐based formula. While isoflavone content of formula varies, it averages 40 μg total isoflavones per gram of formula. A typical newborn or infant fed soy-‐based formula ingests roughly 6-‐11 mg/kg bw isoflavones a day, which is higher than the average for adults consuming a traditional soy based Asian diet (0.3-‐1.2 mg/kg/day) [69]. These infants have plasma isoflavone concentrations between 2.4 and
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6.5 μM, an amount that is up to approximately 5-‐fold higher than the average plasma concentrations reported for Japanese women (1 μM) [67]. An analysis of 240 foods from the UK was done by Kuhnle et al., [70] to determine their phytoestrogen content. Among the foods with the highest genistein content are soybeans and soya flour, with cooked soybeans averaging at 10664 μg/100 g, and soya flour at 62125 μg/100 g. The complete results from Kuhnle et al., [70] can be found in Appendix II.
7.2. Genistein as an endocrine disruptor Figure 6.1 shows that genistein and estradiol are structurally similar. Due to this similarity, genistein can bind both ERα and β, with a preference for the latter. ERβ is largely expressed in the ovary, prostate, lung, GI tract, bladder, central nervous system, and hematopoietic system [64]. Collectively, phytoestrogens are often described as SERMs-‐selective estrogen receptor modulators. This means that in one tissue or during a specific point in development (e.g. puberty), genistein can act as an ER agonist and in a different tissue type or even in the same tissue but during a different point in development, genistein can act as an ER antagonist. Genistein's indiscriminant behavior makes it exceedingly difficult to define how the compound will act in different tissues at different time periods and what the downstream effects might be [64]. Morito et al., [71] in an in vitro competition binding assay demonstrated that genistein binds to ERβ as strongly as 17 β-‐estradiol, although it does not induce transcription to the same extent. In order to induce the same level of transcription as estradiol, 104 times the amount of genistein is needed. In the same study, the authors found that the glycoside form binds poorly to both ERα and β. Glycosides are also poor inducers of transcription. Compared to glycosides, aglycones have a stronger affinity for the ER [71]. Using the uterotrophic assay on ovariectomized mice, Ohta et al., [72] determined the extent of agonism or antagonism of 36 different chemicals, including genistein and genistin, the inactive glycoside. This study was done according to OECD test guidelines No. 440. The group found that both compounds showed agonistic and antagonistic activity and could compete with 17-‐α ethynyl estradiol for binding to ERα. A dose of 220.2 mg/kg bw/day genistein induced a 10% uterotrophic effect, while only 63.5 mg/kg bw/day was enough to inhibit the effects induced by 17-‐α ethynyl estradiol, demonstrating the compounds' abilities to bind to ERα and compete with estradiol [72]. A one-‐generational developmental study was done by Jefferson et al., [73] in which female CD-‐1 mice were subcutaneously injected with genistein dissolved in corn oil for the first 5 days of life at the doses of 0.5, 5, or 50 mg/kg bw/day. These mice were bred to control male mice of the same strain, and pups were sexed and counted when born. The group found that mice exposed to the highest dosage failed to deliver live pups. This study was repeated with a different group of genistein-‐treated mice and the same results were obtained. The authors attributed these results to genistein-‐induced malformations in the ovaries, such as multi-‐oocyte follicles and attenuated cell death of oocytes [73, 74].
7.3. Genistein exposure and immune effects In vitro studies indicate that genistein at high concentrations has effects on various immune system cells. However, these concentrations are not expected to be reached in humans consuming a soy diet, so the physiological relevance of these findings is uncertain. In vitro studies show that genistein can influence cytokine production and secretion, often promoting Th2 immunity [65]. It can activate natural killer (NK) cells of the innate immune system at
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concentrations between 0.1 and 0.5 μM. Genistein has also been shown to inhibit histamine release from basophils in vitro [75]. In vivo studies using animal models tend to have inconsistent results in terms of the effects of genistein and other soy isoflavones on the immune system. In a mouse model of multiple sclerosis, sub-‐cutaneous injection of 200 mg/kg genistein bw/day for 7 days improved the myelin profile in the brain and inhibited the secretion of pro-‐inflammatory cytokines in the brain [76]. In an in vivo study conducted by Yellayi et al., [77] genistein was shown to have a suppressive effect on the immune system of mice. C57BL/6 mice were given subcutaneous injections of genistein at doses of 8, 20, 80, or 200 mg/kg bw genistein once daily for 7 or 21 days. Genistein reduced the weight and size of the thymus in a dose dependent manner, leading to thymic atrophy and rapid apoptosis of thymocytes [77]. Thymic weight in mice injected with 80 mg/kg genistein decreased by 62% after 7 days; mice injected with 80 mg/kg for 21 days had a 73% decrease in thymic weight. An analysis of thymocytes revealed that mice given daily injections of 200 mg/kg genistein for 7 days experienced near total elimination of CD4+/CD8-‐ thymocytes and a severe inhibition of double positive CD4+/CD8+ thymocytes. 80 mg/kg genistein injection suppressed both humoral and cell mediated immunity, reducing specific antibody titers by 80%. This was also seen in a dose-‐dependent manner, as 8 mg/kg genistein reduced antibody titers by 50%. The authors determined that a daily subcutaneous injection of 8 mg/kg bw genistein in mice produces peak plasma blood levels comparable to 4 month old human infants fed soy-‐based infant formula [77]. Asthma patients with a high intake of soy experience better lung function and reduced airway inflammation caused by eosinophils, indicating that isoflavones may be beneficial in allergy [78, 79]. In peanut allergy mouse models, an isoflavone diet containing 1,500 ppm (roughly equivalent to 225 mg/kg bw/day) of genistein and diadzein each suppressed peanut-‐induced anaphylaxis by subduing the degranulation of mast cells, thus inhibiting histamine [65]. C3H/HeJ mice were either fed a soy-‐free diet or one containing 1,500 ppm isoflavones (225 mg/kg genistein bw/day and 225 mg/kg daidzein bw/day) for 2-‐3 weeks. Mice in both groups were subsequently sensitized once weekly with crude peanut solution (10 mg) and cholera toxin (20 μg) for a period of 5 weeks, and then boosted with 50 mg peanut solution and 20 μg cholera toxin for 2 weeks. Mice fed the soy-‐free diet and those fed the isoflavone rich diet were challenged twice in 30-‐minute intervals with 200 mg of peanut oil, and physical symptoms of anaphylaxis (scratching and rubbing around the snout and head, puffiness around eyes and snout, diarrhea, wheezing, labored respiration, no activity after prodding, tremor, convulsions, and death) were scored based on the severity of symptoms. Mice given the soy-‐free diet had severe symptoms of anaphylaxis, while only 60 to 65% of mice in the soy-‐fed diet showed symptoms of anaphylaxis that were significantly less severe in comparison to those fed the soy-‐free diet [80]. The authors contribute the ability of soy isoflavones to reduce allergy symptoms to their suppression of the immune system and ability to inhibit the response of dendritic cells and CD4+ T cells [80]. In vivo rodent studies in which Siebel et al., [81, 82] aimed to demonstrate the protective effects of a phytoestrogen rich diet against inflammatory bowel disease showed that in rats, pre and postnatal dietary exposure to isoflavones did not provide protection against the development of inflammatory bowel disease and in fact enhanced the extent of acute inflammation in rats. Female Wistar rats were divided into two groups: one group receiving a diet (ad libitum) depleted in isoflavones (less than 10 μg/g of genistein and daidzein) and another receiving a diet (ad libitum) rich in isoflavones (240 μg/g genistein and 232 μg/g daidzein). Both groups of females were mated and kept on their respective diets during pregnancy and lactation,
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throughout which the pups were either exposed to a phytoestrogen-‐rich (PRD) or phytoestrogen-‐depleted (PDD) diet. The daily intake of isoflavones was calculated to be approximately 4431 to 4584 μg per animal per day in the phytoestrogen-‐rich diet in comparison to <210 μg isoflavone per day in the phytoestrogen-‐depleted diet. At 11 weeks of age, colitis was induced in the pups by 2,4,6-‐trinitrobenzenesulfonic acid (TNBS). The extent of inflammation was stronger in the PRD animals in comparison to the PDD group. Siebel et al., [81] concluded that further investigations are warranted in order to determine the role of diets rich in phytoestrogens and inflammation. In contrast, Siebel conducted another in vivo study [82] in which male Wistar rats were dosed with 100 mg/kg bw genistein by gavage daily for 14 days. These rats were then exposed to TNBS in order to induce colitis in the same way as in the previous study. In comparison to the controls, genistein-‐treated rats had decreased inflammation in the colon, leading the authors to conclude that genistein exerted beneficial anti-‐inflammatory effects at the exposure dose of 100 mg/kg bw/day in rats. Although not a parameter in the study, genistein-‐exposed rats had lower wet weights of seminal vesicles and prostates than the controls [82]. This study in contrast to the first shows the indiscriminate actions of phytoestrogens. Life-‐long exposure to a diet high in isoflavones increased inflammation in rat models of inflammatory bowel disease, while 14 day exposure to 100 mg/kg bw/day of genistein had the opposite effect and exerted anti-‐inflammatory effects in the same rat models [81, 82].
7.4. Genistein and neuroendocrine effects Losa et al., [83] subcutaneously injected newborn Long Evans rat pups 10 or 1 mg/kg bw genistein every 24 hours for 4 days. The group then sampled sections of the anterioventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) from the brains of exposed and control individuals in order to determine whether neonatal genistein exposure effected the expression of the kisspeptin (KISS) protein family. KISS initiates puberty by stimulating the release of GnRH, and the major populations of KISS neurons are located in the AVPV and ARC of the hypothalamus. Genistein exposed individuals experienced earlier vaginal opening in comparison to controls, with the high dose group achieving puberty before the low dose exposure group. Additionally, neonatal genistein exposure decreased the fiber density of KISS neurons in the AVPV of female rats. Normally, AVPV fiber density increases closer to puberty in females, assisting the characteristic release of GnRH. This was not the case in the 10 mg/kg bw genistein treatment group, who had fiber densities in the AVPV more typical of male brains. The group concluded that in rats, genistein exposure during the neonatal critical period can defeminize the hypothalamus. In the highest dose group, KISS fiber densities persisted into adulthood, indicating that the effects were permanent [83]. Animal behavioral tests for anxiety using the elevated plus maze and memory tests using the radial arm maze have shown that lifelong exposure to dietary phytoestrogens (from conception until adulthood) reduces anxiety behaviors in male and female rats. Lephart et al., [84] fed male and female Long Evans rats four different diets: one containing 0 ≤ 5 ppm isoflavones (0.25 mg/kg bw/day, referred to as the AIN-‐76 diet), a phyto-‐free diet containing 10-‐15 ppm isoflavones (0.5-‐0.75 mg/kg bw/day), a phyto-‐200 diet containing 200 ppm isoflavones (10 mg/kg bw/day) or a phyto-‐600 diet containing 600 ppm isoflavones (30 mg/kg bw/day). Serum genistein levels in rats given the phyto-‐600 diet were 117 ± 5 ng/mL. Those consuming the phyto-‐free diet had serum genistein levels of 6 ± 1 ng/mL. In memory tasks using the radial arm maze, only males and females fed the phyto-‐600 diet with their counterparts fed the phyto-‐free diet were compared to determine the effects of dietary phytoestrogens in learning and memory tasks [84].
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Generally, males consistently outperform females in tasks related to spatial memory. Spatial memory can be experimentally measured in rats by the 8 arm radial maze, in which food deprived animals retrieve a food reward from the different arms and animals are scored by speed, accuracy, and number of errors. In this maze, males fed the phyto-‐free and females fed the phyto-‐600 diet acquired accuracy faster than males and females in the opposite group (males fed the phyto-‐600 and females fed the phyto-‐free were slow to acquire accuracy). This suggests that the effects of dietary isoflavones are sex specific. Lephart et al., [84] then switched diets in some of the young rats from the phyto-‐600 group, feeding them the phyto-‐free diet for 50 days. Thus, males normally consuming a diet rich in phytoestrogens (phyto-‐600 with 30 mg/kg bw/day) began consuming one containing less than 1 mg/kg bw/day of isoflavones. The same was done in females. The rats that had their diets switched were then placed in a 4-‐arm radial maze and their accuracy in finding a food reward was measured (the amount of reference errors made was counted). Rats in the switched diet group were compared to rats that continued with their normal diets. A dietary change from lifelong phytoestrogen consumption (phyto-‐600) to one devoid of phytoestrogens (phyto-‐free) resulted in improvements and greater accuracy of male rats in the maze. Males who had their diets switched committed less reference errors than males fed the phyto-‐600 diets. In females, switching diets from the phyto-‐600 to the phyto-‐free diet led to female rats showing less accurate spatial memory in the radial maze. The researchers hypothesized that the phytoestrogen induced sex-‐reversal of visual spatial memory, leading to enhanced spatial memory in females but compromised spatial memory in males, was due to changes in brain structure and function within the frontal cortex, a brain region correspondingly high in expression of ERβ [84]. Besides measuring learning and memory, Lephart et al., [84] also measured anxiety by using an elevated plus maze (two open and two closed arms with a roof are elevated in the shape of a plus sign). Anxiety was measured by the amount of time animals spent in the closed arms. Females and males from ANF-‐76, phyto-‐free, phyto-‐200, and phyto-‐600 groups were evaluated. A dose-‐dependent reduction in anxiety parameters was observed in males, with males fed the phyto-‐600 diet displaying the lowest anxiety parameters and those fed the ANF-‐76 diet having the highest. Female anxiety behavior displayed the same patterns, although the influence of dietary isoflavones was not as robust as in males [84].
7.5. Conclusion Due to the prevalence of soy and its importance as a major staple diet in certain parts of the world, some humans are likely to be exposed to isoflavones on a daily basis. There is also great individual variation in the absorption and metabolism of genistein, and plasma concentrations can range from 1 μM in Asian consumers to 0.07 μM in Westerners, because to both differences in consumption patterns and individual variations in metabolism [67]. The greatest exposure occurs in infants fed soy-‐based infant formula, with formula containing an average of 40 μg isoflavones content per gram of formula [69]. Infants consuming soy-‐based formula typically have average plasma isoflavone concentrations between 2.4 and 6.5 μM [67]. It is known that genistein binds to and competes with 17β-‐estradiol for binding to the ER [71]. Due to its properties as a SERM, it is difficult to predict the behavior of genistein or the consequences of exposure. Animal studies examining immune effects resulting from genistein exposure often demonstrate that genistein has suppressive effects on the immune system, which are beneficial in mediating the effects of inflammation or allergic reactions. However, at least one study has indicated that animals fed a lifelong diet containing isoflavones may actually experience increased inflammation during colitis in comparison to animals not consuming isoflavones [81, 82]. In behavioral and anxiety tests, phytoestrogens appear to be beneficial in decreasing anxiety in rats, with a greater anxiety-‐relieving properties in males than in females.
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Likewise, isoflavones seem to have a sexually dimorphic influence on visual and spatial memory, enhancing spatial memory in females while inhibiting it in males [84]. More studies should be undertaken in order to determine the effects of dietary phytoestrogen exposure on neuroendocrine and immune parameters. Monitoring or cohort studies can be done on infants consuming soy-‐based formula in order to better determine whether high levels of plasma isoflavones at an early life stage can have effects on the endocrine, nervous, or immune systems later in life.
8. Triazole fungicides The third compound examined is tebuconazole, part of the group of triazole fungicides. Triazoles belong to the class of conazole pesticides. They are used worldwide for protection of cereal grain, vegetables, fruits, and flower production. Triazoles are also used as pharmaceuticals for the treatment of human fungal infections, such as vaginal mycosis in pregnant women and thrush in infants [85]. Figure 8.1 illustrates the structure of tebuconazole.
Figure 8.1. Structure of tebuconazole. Conazoles inhibit the enzyme lanosterol 14-‐α-‐demethylase (also called CYP51), which regulates the synthesis of ergosterol, an essential component of the fungal cell wall [85, 86]. The inhibition of ergosterol causes the fungus to grow abnormally, resulting in death. Evidence indicates that triazoles may also bind to human cytochrome P450 (CYP) enzymes, including aromatase (CYP19), the enzyme that converts testosterone into estradiol, thus giving triazoles their status as endocrine disruptors [85]. In studies where animals have been exposed to triazoles, diverse effects have been observed including craniofacial and brain malformations, variations in the urinary tract, and decreased fetal weight. Triazoles have been implicated in reproductive toxicity as they have been shown to impair fertility, prolong gestation, and reduce pup survival and litter weights [77]. Triazoles are also hepatotoxic and induce liver effects ranging from enzyme induction to inflammation and necrosis, with possible liver outcomes contributing to tumors in thyroid follicular cells [87]. Tebuconazole is rapidly absorbed and widely distributed to different tissues in mammals, with high concentration residues found in kidneys and liver. The compound does not have the potential to accumulate and is excreted extensively, mostly in feces [87]. Tebuconazole is a moderate acutely toxic substance in rats, with an LD50 of 1700 mg/kg bw. In sub chronic to chronic toxicity tests (90 days and 1 year) carried out in rats, rabbits, and dogs, the lowest relevant NOAEL was found to be 3 mg/kg bw/day due to hypertrophy in the zona fasciculate of the adrenals in dogs exposed for 1 year. Based on this NOAEL (3 mg/kg bw/day) and an uncertainty factor of 100, the ADI of tebuconazole was established to be 0.03 mg/kg bw/day [87]. Tebuconazole is extensively metabolized into triazole alanine in wheat grains and peanut kernels. In all other plant parts, tebuconazole metabolism occurs at a very low extent. When it does occur, the metabolites are 1,2,4-‐triazole and triazole acetic acid [87]. Depending on the
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commodity, consumers can be exposed to both the parent compound and its metabolites. Tebuconazole’s metabolites may also have effects on the endocrine system separate from the effects of the parent compound. The ADI for the metabolites, which are also produced by other substances belonging to the triazole group, are as follows: triazole alanine at 0.1 mg/kg bw/day; 1,2,4-‐triazole at 0.02 mg/kg bw/day; triazole acetic acid at 0.02 mg/kg bw/day [87].
8.1. Exposure analysis Taking into account chronic toxicity, the highest theoretical maximum daily intake (a measure of chronic exposure) of tebuconazole was calculated to be 16.8% of the ADI (or 5.04 µg/kg bw/day) in the WHO Cluster B group, using the EFSA pesticide residue intake model [88]. More extensive risk and consumer assessment needs to be done that also takes into consideration the exposure to tebuconazole's metabolites [87].
8.2. Tebuconazole as an endocrine disruptor Kjaerstad et al., [85] found that tebuconazole was anti-‐estrogenic, inhibiting the estrogen-‐induced proliferation of MCF-‐7 cells at a concentration of 1.6 μM in vitro. Tebuconazole was also an AR antagonist at a concentration of 3.1 μM in vitro. Also in vitro, the concentrations of progesterone increased at doses from 3 to 10 μM, while concentrations of testosterone and estradiol decreased between the doses 3 and 30 μM. Kjaerstad suggests that the effects of tebuconazole on hormone levels is possibly due to the inhibition of the enzyme that converts progesterone into testosterone, CYP17 [85]. In vivo studies have shown effects in animals exposed to tebuconazole at doses higher than the NOAEL of 3 mg/kg bw/day [89, 90]. For instance, in vivo exposure of pregnant rats to tebuconazole at the LOAEL (10 mg/kg bw/day) leads to F1 generation females with reduced uterus weights and males with reduced epididymis weights [89, 90]. Pregnant rats gavaged with 100 mg tebuconazole/kg bw/day from gestational day 7 to postnatal day 16 gained less weight and had longer gestational periods than controls. Additionally, postnatal death of the pups occurred in this dose group, which was not seen in the control group [90]. In a different study [89], a greater number of dead pups were seen in litters in which dams were orally given 60 mg tebuconazole/kg bw/day, an effect that was not seen in the lower dose groups of 0, 6, or 20 mg/kg bw/day [89]. Other effects of tebuconazole exposure in utero include the feminization of male offspring; dams exposed to doses of 0, 50, or 100 mg/kg bw/day via gavage between GD 7 and 16 gave birth to male pups with an increased number of nipples in both the 50 and 100 mg/kg bw/day dose groups. Male pups of dams given 100 mg/kg bw/day in utero had decreased serum testosterone concentrations during gestational day 21. Males of dams in the low dose group (50 mg/kg bw/day), but not the high dose group, had increases in serum progesterone levels. Female fetuses exposed in utero to 50 or 100 mg tebuconazole/kg bw had increased anogenital distances at birth [90]. The dams dosed with 50 and 100 mg/kg bw/day experienced a sevenfold increase in plasma progesterone levels, which is consistent with the in vitro studies performed by Kjaerstad et al. [85].
8.3. Tebuconazole exposure and immune effects In general, there is a lack of studies detailing the effects of tebuconazole on the immune system. One study by Moser et al., [89] examines immune, nervous, and reproductive effects of tebuconazole on pregnant dams and their pups. Pregnant dams were fed tebuconazole at doses of 0, 6, 20, or 60 mg/kg bw/day from gestational day 14 until postnatal day 7. The F1 generation was subsequently fed the same doses from postnatal day (PND) 7 until PND 42. The exposure period was chosen to mimic the exposure period until puberty. Both F0 and F1 generation
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males and females were assessed for the immunological, neurological, and reproductive effects (section 8.2) of tebuconazole exposure. Immunological assessment included measurement of the weights of the spleen and thymus, analysis of the B, T, and natural killer cell response in the spleen of F1 rats exposed to three different mitogens, and a measurement of cell proliferation using the plague forming assay. Tebuconazole was found to decrease spleen weight in F1 males, at 60 mg/kg bw/day, with no other effects seen in the parameters tested [89].
8.4. Tebuconazole exposure and neuroendocrine effects Tebuconazole also inhibits voltage gated calcium channels upon depolarization, leading to reductions in calcium influx [91]. Rat PC12 cells (a model of mature dopaminergic neurons) were exposed to six azole fungicides, including tebuconazole, in order to determine their effects on basal Ca2+ and intracellular Ca2+ after depolarization. The in vitro study found that tebuconazole in combination with imazalil, flusilazole, triadimefon, and cyproconazole inhibits Ca2+ influx into PC12 cells after depolarization in a concentration-‐dependent manner [91]. Because calcium is essential in neuronal signaling, inhibition of calcium channels during depolarization may reduce neurotransmission in dopaminergic neurons [91]. From the study by Moser et al., [89], a neurotoxicological assessment was carried out including functional observational battery (FOB-‐a series of operational and manipulative tests designed to assess the neurological integrity of the test subject), automated measure of motor activity, passive avoidance (to test retention memory), and the Morris water maze (to test spatial and working memory). F1 rats were tested twice: once during PND 49 and 50, and again during PND 70 and 71. While the researchers did not see significant effects of tebuconazole exposure in most of the assessments used, it was found to significantly alter spatial memory, as male and female rats in the highest dose group (60 mg/kg day) learned the position of the platform in the Morris water maze at a slower rate in comparison to controls. Females displayed delayed learning during the first testing battery only (PND 49 and 50) while males were slower than controls during both testing dates (PND 40 and 50 and again PND 70 and 71) [89]. Besides tebuconazole, other triazole fungicides have adverse effects on the CNS. For instance, triadimefon and triadimenol are psychomotor stimulants in rats, leading to hyperactivity and similar behaviors to those seen in rats given amphetamine. Male Long-‐Evans rats fed a single oral dose (from 50 to 400 mg/kg of both compounds; exact dose not specified by the authors) dissolved in corn oil showed hyperactive behavior and significant increases in activity measured by behaviors such as excessive grooming, head bobbing, sniffing and rearing at doses greater than 50 mg/kg. In this study, tebuconazole was also given to rats at doses from 50-‐2000 mg/kg bw, with no behavioral outcomes. The author speculates that the behavioral effects of triadimefon and triadimenol are due to the structure of both compounds. Triadimefon and triadimenol have an ether oxygen, which is replaced by a carbon moiety in other triazoles. The structure may explain why these specific triazoles out of a total of 16 tested produced hyperactive behavior. Both compounds are thought to inhibit or decrease the reuptake of dopamine from the synapse in nerve terminals, leading to hyperactive behaviors [92]. Evidence from a zebrafish study by Liu et al., [93] indicates that triazoles may influence signaling in the HPT axis. Exposure of zebrafish embryos to triadimefon induced an upregulation of mRNA expression for thyroid hormone T4 but decreased expression of thyroid hormone receptor beta (THR-‐β). Additionally, expression of the gene dio1, which codes for the deiodinase that converts T4 into T3, was decreased [93].
8.5. Conclusion According to estimation of chronic exposure, the highest theoretical maximum daily intake of tebuconazole is expected to be approximately 5 μg/kg bw/day [88], which is below the ADI of
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30 µg/kg bw/day. Evidence of the endocrine disrupting properties of tebuconazole includes its ability to inhibit the proliferation of MCF7 cells and antagonize the AR in vitro [85]. In vivo animal studies have shown negative effects of exposure at doses higher than the NOAEL of 3 mg/kg bw/day. These effects include reduced organ weights and increased rates of death in pups of exposed dams [89, 90]. There is a general lack of information detailing immune effects of chronic exposure to tebuconazole, but one study [89] was able to demonstrate that oral exposure of tebuconazole at 60 mg/kg bw/day decreased spleen weights of males exposed in utero. An in vitro study by Heusinkveld et al., [91] showed that tebuconazole in combination with four other triazole fungicides inhibits calcium influx into PC12 neurons during depolarization in a concentration-‐dependent manner. Because calcium is essential in neuronal signaling, inhibition of Ca2+ channels may reduce neurotransmission in dopaminergic neurons. To date, Heusinkveld's in vitro results have not been explored in vivo. However, an in vivo study by Crofton [92] exploring neurotoxic effects of triazoles found that rats ingesting at least 50 mg/kg bw daily of two other triazole fungicides, triadimefon and triadimenol, exhibited hyperactive behavior. While Crofton suggested that the development of hyperactive behavior was due to a decrease in the reuptake of dopamine from synaptic nerve terminals, and not due to calcium inhibition, this study supports the notion that triazoles have effects in the CNS. Based on the studies reviewed in this report, the estimated exposure of tebuconazole does not indicate that it induces endocrine disruption in humans, but the combined exposure of all triazoles and their metabolites must be further investigated.
9. Discussion The studies mentioned in this thesis have demonstrated that the investigated compounds have endocrine disrupting properties. However, the critical question of whether exposure via food to the endocrine disrupting compounds BHA, genistein, and tebuconazole contributes to adverse effects in the nervous and immune systems of humans has yet to be answered. Based on the scientific literature presented in this report, it is clear that these three compounds certainly have the potential to exert adverse effects in both systems. Perhaps this question cannot hitherto be answered within the current state of the science due to a lack of studies as well as inadequate screening and testing methods specifically designed to accommodate immune and nervous system endpoints. The validated Testing Guidelines (TG) used by the OECD tailored specifically to endocrine effects are outlined in the Conceptual Framework included in Appendix I. As of 2012, when the framework was last revised, only one TG (OECD TG 426) addressed developmental neurotoxicity [94] and there are no TGs for immune modalities listed in the Conceptual Framework document. In terms of the compounds BHA, genistein, and tebuconazole, all studies (in vitro and in vivo alike) have indicated that exposure or contact with the particular compound in some way may have either direct or indirect effects on the immune and nervous system. Effects of the compounds on both systems are summarized in the conclusion sections 6.5, 7.5, and 8.5. Striking findings from in vitro studies of BHA and tebuconazole exposure that should be explored further are the abilities of both compounds to modify levels of Ca2+ in exposed cells [57, 60, 91]. For instance, BHA has the potential to modify the lymphocytes of the immune system by influencing intracellular calcium concentrations. Calcium oscillations in lymphocytes act as signals that activate B and T cells and lead to the synthesis of cytokines that can subsequently have effects on inflammation, growth, or activation of cells [58]. The ability of BHA to modify
36
Ca2+ levels was observed in two in vitro studies (53, 55). Interestingly, one study demonstrated that BHA exposure in human umbilical cord endothelial cells, rat cardiomyocytes, and rat pituitary cells lead to increases in the levels of intracellular calcium in a dose-‐dependent manner [59]. The other study failed to find an increase of intracellular Ca2+ in thymocytes and splenocytes exposed to BHA in vitro. In fact, BHA exposure prevented an increase in calcium in exposed cells [57]. As both studies were conducted in vitro, it is unclear what the results could mean in terms of a real-‐world exposure scenario. To my knowledge, there have not yet been any in vivo studies of BHA exposure on intracellular calcium levels in mammals in the scientific literature. In addition to immune effects, changes in intracellular calcium levels can implicate neuronal function, as Ca2+ is essential in the propagation of an action potential [91]. Tebuconazole in combination with several other conazole fungicides (imazalil, flusilazole, triadimefon, and cyproconazole) inhibits voltage gated calcium channels in dopaminergic neurons (PC12 cells) upon depolarization in vitro. A greater degree of inhibition was seen at higher concentrations of fungicides, indicating that the inhibition occurs in a concentration-‐dependent manner. Heusinkveld et al., [91] also pointed out that the inhibitory effects of the compounds on Ca2+ influx were additive. Exposure of PC12 cells to a single fungicide did not affect basal levels of calcium; only when PC12 cells were exposed to a mixture was basal Ca2+ inhibited [91]. Similar to the studies previously described for BHA, the results from Heusinkveld's study on tebuconazole are not enough to explain the possible in vivo effects of tebuconazole exposure on intracellular calcium levels. More studies need to be done to elucidate the outcomes of calcium modulation and what that modulation means for the nervous and immune systems. Calcium levels have not traditionally been considered an endpoint conducive to screening and testing for EDCs. As a second messenger molecule, the role of Ca2+ in the propagation of intra and extra cellular signaling has been underappreciated. In the nervous system, Ca2+ is responsible for dopaminergic neurotransmission, gene transcription, neurodegeneration, and neurodevelopment [91]. In the immune system, Ca2+ is responsible for the mobility of lymphocytes, T-‐cell mediated toxicity, cell differentiation, and effector functions [95]. Calcium levels in the blood are controlled by the endocrine system; namely, parathyroid hormones and vitamin D increase the concentration of calcium in the blood, while calcitonin reduces blood calcium levels [96]. While these hormones are not discussed in any detail in this report, their roles as targets of EDCs should be considered in future investigations. Since BHA and tebuconazole can both modify intracellular calcium levels, it could be interesting to note whether they can also affect these hormones, leading to changes in the levels of calcium in the blood. Modifications to calcium homeostasis can have effects on intracellular signaling, ultimately leading to downstream or indirect consequences on programming or the set-‐up of homeostatic pathways essential to the proper function of the immune and nervous systems. While the studies included in this report detailing the effects of exposure to genistein in the diet have not examined the role of the compound’s effects on calcium, some studies have indicated that genistein modifies the immune response and can play a role in immune suppression by increasing or decreasing inflammation [65, 80-‐82]. Lymphocytes and inflammatory cytokines released from lymphocytes and other immune cells are responsive to changes in intracellular calcium levels. For instance, prolonged increases in Ca2+ levels in B and T cells lead to changes in DNA expression in these cells and contribute to the transcription of inflammatory cytokines [95]. Thus, modification of calcium signaling can be an important pathway with which soy isoflavones exert their effects on the immune system and should be further explored. Nervous system effects of BHA are not well documented and few studies have extensively investigated the consequences of BHA exposure on neurodevelopmental endpoints. The study included in this report noted BHA-‐exposed rats exhibited a delay in the auditory startle response compared to control rats by a day and a half [62]. It is unclear what these results
37
indicate, as BHA in this study did not induce behavioral toxicity in rats even at the highest exposure level of 0.80 g/kg bw/day (0.50% wet weight of the diet). There were no studies on tebuconazole exposure and neurological effects, although a study by Heusinkveld et al., [91] noted that in vitro exposure of neurons to a mixture of tebuconazole and several other azole fungicides inhibited voltage gated Ca2+ channels upon depolarization. Genistein exposure does have effects on the nervous system of mammals that warrant additional investigation. Studies on neonatal exposure to genistein indicate that the compound may have effects on brain regions associated with male and female behavior, such as the hypothalamus [83]. For instance, subcutaneous injection of 10 mg/kg genistein bw/day for 4 days lead to female rats with a decreased density of neurons in the AVPV of the hypothalamus, leading to brains that resembled those of males. The fiber densities of the affected neurons in the AVPV persisted into adulthood, indicating that genistein's de-‐feminizing effects were permanent [83]. Plasma levels of isoflavones corresponding to the concentration of 10 mg/kg bw/day were not measured, so it is unclear whether the concentrations the animals were exposed to pose a threat to normal development of the hypothalamus in animals consuming a standard diet containing isoflavones. Humans, in any case, consume isoflavones at concentrations lower than 10 mg/kg bw/day, with Asians averaging between 0.3 and 1.2 mg/kg bw/day [67]. Infants fed soy-‐based formula are the highest consumers of soy isoflavones, ingesting roughly 6-‐11 mg/kg bw/day of isoflavones [67]. These doses are comparable to the concentrations given to rats in the study by Losa et al. [83]. A critical difference, however, is that infants consume genistein orally, and the animals in Losa’s study were administered genistein via subcutaneous injection. Genistein undergoes metabolism in the infant GI tract before reaching blood plasma, while genistein in rats injected subcutaneously skips metabolism in the GI tract, leading to a different plasma concentration in rats. Additionally, genistein typically exists in its inactive, glycoside form. Ingesting genistin glycoside actually activates the compound as salivary enzymes and bacterial glycosidases convert genistin into the aglycone form (genistein) in the gut. The activity of activated genistein is short-‐lived and restricted to the gut, as it is immediately conjugated with glucuronides, sulfates, or acetates in intestinal epithelial cells [80]. Is it likely that exposure to BHA, tebuconazole, and genistein at the current levels can lead to adverse effects in humans? In all the studies mentioned in this report, there have not been any documented adverse effects in animals exposed to BHA or tebuconazole at levels lower than the NOAEL. The NOAEL for BHA was 100 mg/kg bw/day based on growth retardation, increased mortality, and behavioral effects in rat pups. Due to this NOAEL and an uncertainty factor of 100, the ADI for human exposure was set to 1.0 mg/kg bw/day by EFSA. Exposure to BHA is unlikely to occur at levels higher than the ADI. According to EFSA and based on the estimated maximum permissible levels (MPLs) of BHA in food, country-‐specific data for Sweden (Table 6.1) indicates a lower mean level of exposure in comparison to other European countries. However, exposure at levels higher than the ADI may be a reality in individuals who are both exposed via food and who use plastic storage containers to keep their foodstuffs. EFSA estimated that children and toddlers who eat 1 kg of food packed in BHA-‐contained plastic at the maximum allowed levels can actually be exposed to BHA at levels higher than the ADI [51] (children: 1.3 mg/kg bw/day; toddlers: 2.5 mg/kg bw/day). Because EFSA's calculations of exposure are based on MPLs and not on actual values of BHA content in food, this exposure estimate is likely to be conservative, meaning humans are not likely to be exposed to BHA at doses which can lead to adverse health effects. However, a thorough exposure assessment should be done in order to verify that levels of BHA in food are actually below the MPLs as they should be, and that humans are indeed exposed in a similar fashion as estimated by EFSA. Tebuconazole exposure is estimated to be maximally about 17% of the ADI of 0.03 mg/kg bw/day, which means that human exposure is well under the ADI [88]. Additionally, the studies
38
referenced in this report that indicated adverse effects as a result of in vivo tebuconazole exposure occurred at exposure levels higher than the NOAEL of 3 mg/kg bw/day [89, 90]. As mentioned earlier, additional in vivo testing could be done to determine whether tebuconazole exposure at doses lower than or close to the NOAEL can lead to modifications of intracellular calcium levels in order to determine whether tebuconazole can implicate important signaling pathways. Exposure to tebuconazole alone does not indicate that adverse effects on the endocrine system will occur, especially if exposure is below the NOAEL. However, the cumulative exposure of several triazoles and their metabolites may lead to adverse effects. This is something that needs to be further investigated. There is no ADI or NOAEL for genistein. In a search of the scientific literature, no studies were found documenting adverse effects of phytoestrogen or isoflavones exposure on human health. Contrarily, the isoflavones genistein and daidzein are of great interest primarily due to their prevalence in food and their bioactivity. These compounds have received much attention because of their potentially beneficial effects on cancer, cardiovascular disease, osteoporosis, menopausal symptoms, male infertility, obesity, and type II diabetes [70]. Benefits to these conditions are credited to the fact that the highest consumption of isoflavones occurs in Asian countries, where rates of certain cancers and other hormonal conditions are far lower than in the West [35, 84]. In an EFSA opinion on the substantiation of health claims related to soy isoflavones, EFSA concluded that there was insufficient evidence to support that exposure to soy isoflavones had beneficial effects on the maintenance of bone mineral density in post-‐menopausal women [97, 98], the reduction of vasomotor symptoms associated with menopause, protection of DNA, proteins, and lipids from oxidative damage, or the maintenance of normal blood LDL-‐cholesterol concentrations [97, 98]. Despite the absence of an ADI or a NOAEL, it seems that genistein exposure in newborn rat pups alters brain physiology in regions associated with timing of puberty [83]. Results from this study are difficult to extrapolate in terms of human exposure, as rat pups were injected subcutaneously and humans are exposed to genistein orally. Further investigation is warranted as genistein can be transported across the placenta and bind to ER in the underdeveloped brain, as the blood brain barrier (BBB) is immature in utero and immediately after birth. Besides the observation that inhibition of intracellular calcium was a common feature of exposure to BHA and tebuconazole, and modification of the immune response common in studies involving genistein exposure, effects of BHA, tebuconazole, and genistein on Ca2+ should be investigated further, both with controlled in vitro and in vivo studies. Modification of calcium levels can have downstream consequences on cellular signaling pathways, indirectly affecting endocrine function or developmental programming and leading to adverse effects. Another important aspect of endocrine disruption is the impact of chemicals on the epigenome. Current screening and testing methods do not yet take into consideration the epigenetic effects of chemical exposure. Epigenetic alterations, while leaving the DNA intact, can have effects on the expression of proteins and the availability of the transcription machinery to access specific genes or regions of the DNA, transcribing important genes. Epigenetic effects are briefly mentioned in the OECD DRP on novel in vitro and in vivo testing methods [25], but studies utilizing epigenetic endpoints are lacking for BHA, genistein, and tebuconazole. Screening or testing these compounds for their impact on the epigenome could be something to do in the future. Perhaps epigenetic effects may elucidate whether these compounds impact developmental programming. Effects on the epigenome may also shed some light on the broader question of the relationship between the increase in chronic, non-‐infectious diseases, such as neurodevelopmental disorders, and their occurrence in highly industrialized countries where chemical exposure is the norm [1].
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10. Conclusion A review of the scientific literature was completed in order to determine the issues surrounding endocrine disruptors and to discover possible contributions of the antioxidant butylated hydroxyanisole (BHA), the phytoestrogen genistein, and the triazole fungicide tebuconazole on the development and function of the immune and nervous systems. The literature revealed that all three compounds impacted different aspects of both body systems, with BHA and tebuconazole sharing the common mechanisms of intracellular calcium inhibition in several in vitro studies. This is an important finding, as calcium has profound effects on the nervous system, acts as a second messenger in cell signaling, and can determine the cytokine expression profile of B and T lymphocytes. Additional research should be done in order to more thoroughly explore this observation and the relationship between modulation of intracellular calcium levels and endocrine disruption. While there were no studies linking genistein exposure to calcium inhibition, several in vivo studies demonstrated that genistein was able to modify the immune system of rats. Its role on the nervous system is not yet clear. Future research in the area of the effects of endocrine disrupting compounds (EDCs) should be focused on Testing Guidelines for outcomes specific to the immune and nervous systems, as screening and testing in these areas is deficient. Additional research should focus on the effects of EDCs on calcium homeostasis, as this could also be an area where multiple indirect effects occur as a result of disruption, leading to adverse effects on endocrine-‐mediated pathways.
11. Acknowledgements I would like to thank my thesis supervisors at Livsmedelsverket, Anneli Widenfalk and Kettil Svensson, for their constant support, proofreading, and answering of my many questions. Our Monday afternoon meetings were something I very much looked forward to, as I received the greatest inspiration after our meetings. Additional thanks go out to everybody at Livsmedelsverket who went out of their way to make me feel welcome and who fixed all the little things for me, such as an office space and help with finding articles. I would also like to thank my supervisor at Uppsala University, Jan Örberg. Although I was a bit optimistic with the timing of my deadline, you did not lose patience with me and provided very helpful insight and comments on my drafts. Thank you for being such a wonderful human being and an inspirational professor during my time at Uppsala.
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Appendix I
OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters (as revised in 2012)
The OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters (as revised in 2012) lists the OECD Test Guidelines and standardized test methods available, under development or proposed that can be used to evaluate chemicals for endocrine disruption. The Conceptual Framework is intended to provide a guide to the tests available which can provide information for endocrine disrupters’ assessment but is not intended to be a testing strategy. Furthermore, this Conceptual Framework does not include evaluation of exposure; however this should be included when deciding whether further testing is needed. Further information regarding the use and interpretation of these tests is available in Guidance Document No. 150
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Notes to the OECD Revised Conceptual Framework Note 1: Entering at all levels and exiting at all levels is possible and depends upon the nature of existing information and needs for testing and assessment. Note 2: The assessment of each chemical should be made on a case by case basis, taking into account all available information. Note 3: The framework should not be considered as all inclusive at the present time. At levels 2, 3, 4 and 5 it includes assays that are either available or for which validation is under way. With respect to the latter, these are provisionally included.
