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Adrenocortical endocrine disruption
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Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
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Review
Adrenocortical endocrine disruption Philip W. Harvey * Toxicology Department, Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2014 Received in revised form 12 September 2014 Accepted 14 October 2014 Available online xxx
The adrenal has been neglected in endocrine disruption regulatory testing strategy. The adrenal is a vital organ, adrenocortical insufficiency is recognised in life threatening “adrenal crises” and Addison’s disease, and the consequences of off-target toxicological inhibition of adrenocortical steroidogenesis is well recognised in clinical medicine, where drugs such as aminoglutethimide and etomidate killed patients via unrecognised inhibition of adrenocortical steroidogenic enzymes (e.g. CYP11B1) along the cortisol and aldosterone pathways. The consequences of adrenocortical dysfunction during early development are also recognised in the congenital salt wasting and adrenogenital syndromes presenting neonatally, yet despite a remit to focus on developmental and reproductive toxicity mechanisms of endocrine disruption by many regulatory agencies (USEPA EDSTAC; REACH) the assessment of adrenocortical function has largely been ignored. Further, every step in the adrenocortical steroidogenic pathway (ACTH receptor, StAR, CYP’s 11A1, 17, 21, 11B1, 11B2, and 3-hydroxysteroid dehydrogenase D4,5 isomerase) is known to be a potential target with multiple examples of chemicals inhibiting these targets. Many of these chemicals have been detected in human and wildlife tissues. This raises the question of whether exposure to low level environmental chemicals may be affecting adrenocortical function. This review examines the omission of adrenocortical testing in the current regulatory frameworks; the characteristics that make the adrenal cortex particularly vulnerable to toxic insult; chemicals and their toxicological targets within the adrenocortical steroidogenic pathways; the typical manifestations of adrenocortical toxicity (e.g. human iatrogenically induced pharmacotoxicological adrenal insufficiency, manifestations in typical mammalian regulatory general toxicology studies, manifestations in wildlife) and models of adrenocortical functional assessment. The utility of the in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled ‘Endocrine disruptors and steroids’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Adrenal cortex Adrenocortical insufficiency Adrenal toxicity Adrenal hypertrophy H295R cell line Steroidogenesis Stress Toxicity
Contents 1. 2. 3. 4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of the adrenal gland conveying vulnerability to toxicity . . . . . . . . . . . . . . . . Chemicals and toxicological targets within the steroidogenic pathway of the adrenal cortex Manifestation and evidence of adrenocortical toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human clinical experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Regulatory toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Wildlife . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Models of adrenocortical assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo approaches to adrenocortical functional evaluation . . . . . . . . . . . . . . . . . . . . . 5.1.
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* Tel.: +44 1423848421. E-mail address: [email protected] (P.W. Harvey). http://dx.doi.org/10.1016/j.jsbmb.2014.10.009 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: P.W. Harvey, Adrenocortical endocrine disruption, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/ 10.1016/j.jsbmb.2014.10.009
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6.
5.2. In vitro adrenocortical model: H295R cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction The main focus of endocrine disruption interest has centred on reproductive and developmental endpoints, largely dictated by a regulatory framework for the testing and registration of chemicals arising from the United States Environmental Protection Agencies (USEPA) Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations for specific studies enforced in the mid 1990s [1] and later adopted by other regulatory initiatives. Specific evaluation of compounds for (anti) estrogenic and (anti-) androgenic properties in a range of in vitro and in vivo studies, in a tiered approach triggered by findings, forms the core of the testing requirements and the protocols are followed by regulatory toxicology registrants and the research community alike, to a greater or lesser degree of robustness. Recognition that some developmental processes, particularly in wildlife species, are controlled by thyroid function led to the incorporation of some tests to examine this gland. In terms of an overall strategy for endocrine disruption this regulatory framework is lacking because it only focuses on reproductive and developmental endpoints and ignores the integrated function of the endocrine system as a whole and particularly the adrenal gland [2,3] although for chemical registrations there will usually be a range of chronic toxicity and carcinogenicity studies with full microscopic histopathological evaluation of all organs and tissues including the endocrine system (pituitary, thyroid, parathyroid, pancreas, adrenal, ovary and testis) and endocrine target tissues (e.g. mammary, uterus, prostate, etc.). The problem arises in how to interpret toxicopathological changes in these non reproductive endocrine organs, particularly the adrenal as the most common toxicological target in the endocrine system [4], without additional supportive mechanistic studies. A particular weakness has centred on the interpretation of the most commonly observed change in the adrenal namely adrenal (adrenocortical) hypertrophy. Adrenal hypertrophy (increase in gland weight or increase in zone width or cell size microscopically) is often dismissed as stress in the absence of any corroboratory evidence of retained adrenocortical functionality, which is ignorant of the possibility of toxicity in the steroidogenic pathway resulting in glucocorticoid insufficiency. In such cases, glucocorticoid insufficiency results in the loss of feedback regulation to hypothalamus and pituitary and in compensatory over secretion of adrenocorticotrophic hormone (ACTH) and this stimulation promotes growth/ hypertrophy in the zona fasciculata and visibly larger and heavier adrenals [5]. Whilst the current EDSTAC related screening strategies have ignored the adrenal (surprisingly given its role as a vital organ and involvement in development processes, where human conditions such as congenital adrenogenital syndrome or salt wasting conditions expressing neonatally are evidence of the consequences of developmental adrenocortical dysfunction) its importance has been recently recognised by the United States Food and Drug Administration (USFDA) guidance on Endocrine Disruption Potential of Drugs: Non-Clinical Guidance [6]. These draft recommendations recognise the need for enzyme assays including CYP11A1, 11B1, 11B2, 17A1 and 21A1 which encompass the steroidogenic pathways relevant for adrenocortical glucocorticoid and mineralocorticoid metabolism. These recommendations also suggest that endocrine function studies (e.g. measuring hormone levels) are employed to elucidate mechanisms of
histopathological changes seen in endocrine organs specifically including the adrenal, and recognise the adrenal/glucocorticoid action in early life developmental processes (e.g. bone growth). Essentially, these draft guidelines propose the need for full supportive endocrine evaluation when indicated by any changes (usually microscopic histopathology in the non clinical study protocols used) detected in the endocrine system and delineate approaches for expected changes (e.g. drug pharmacological action) from the unexpected. Indeed, the experience of the unexpected adrenocortical toxicity of drugs reaching the market such as aminoglutethimide and etomidate [2,3,5,7] has demonstrated that off target adrenocortical steroidogenic enzyme inhibition can produce fatal adrenal insufficiency in patients. In turn, because of commonality of mechanisms, this raises the issue of whether low level environmental chemical exposures may be impairing adrenocortical function in humans and wildlife. Aquatic wildlife are often considered sentinel species (they are continuously exposed to chemicals in water) and evidence is emerging of adrenocortical endocrine disruption in fish [8] and dolphins [9] as measured by impaired cortisol production, and in the dolphin study impaired aldosterone production was also reported. This brief review will now outline the properties of the adrenal cortex that impart vulnerability to toxic insult, the range of chemicals known to affect the adrenal and their targets within the steroidogenic pathway, models of adrenocortical assessment including species limitations and in vitro test systems (H295R adrenocortical cell line), and approaches to distinguishing stress related changes from toxicity when the common finding of adrenal hypertrophy is observed. Full reviews of adrenal toxicity and toxicologic pathology can be found elsewhere [10–13]. 2. Characteristics of the adrenal gland conveying vulnerability to toxicity The adrenal gland is the most commonly affected endocrine organ in toxicology studies [4]. Part of the reason for this is the central physiological role of the adrenal in the stress response, where toxic insult from the administration of drugs and chemicals administered at the maximum tolerated dose is often stressful and activates the hypothalamo–pituitary–adrenocortical (HPA) axis to increase ACTH secretion. The characteristic changes consistent with increased ACTH stimulation of the adrenal gland are increased size and weight macroscopicallyandincrease in zona fasciculata thicknessand/orcell size microscopically, comprising the typical actions after which this trophic hormone was named. The mechanism of HPA activation and the stress response to environmental insults, including toxicity, is a survival mechanism designed to modify the animals ability to cope physiologically and metabolically to adverse conditions primarily effected by increased glucocorticoid exposure and whilst glucocorticoids have a number of actions (e.g. glucose utilisation and diversion of metabolic resources) it is considered that actions of these steroids (cortisol or corticosterone depending on species) in quenching inflammation which could otherwise overwhelm the organism is the vital action of this system [14]. Indeed, it is the same physiological and anatomical attributes of the HPA axis that are required to effectively modulate the stress response (e.g. rich blood supply to rapidly distribute steroids systemically) that also convey vulnerability of the adrenal to exposure to toxicants and in turn toxic insult. Such properties have been identified [7,12] and are listed below:
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High vascularity - Disproportionately large blood volume received per unit mass of
3
line (discussed later) and documents that almost every step in adrenocortical steroidogenesis is known to be disrupted by a chemical and this is discussed in Section 3.
adrenal tissue ensuring exposures to toxicants Lipophilicity due to rich cholesterol and steroid content
3. Chemicals and toxicological targets within the steroidogenic pathway of the adrenal cortex
favouring local deposition of lipophilic compounds High content of unsaturated fatty acids in adrenocortical cell
membranes susceptible to damage by lipid peroxidation both directly (via parent compound or its metabolites) and indirectly (via generation of reactive species such as free radicals) High content of cytochrome P450 (CYP) enzymes present in the adrenal cortex which can produce: - Reactive parent compound metabolites of toxicants that then
mediate toxicity - Hydroxylation reactions that may generate free radicals which
then damage adrenocortical cells and membrane (as above) In addition, the adrenal cortex is the final effector organ in a chain of events and factors elsewhere in the HPA system, and peripherally, may influence adrenocortical function, such as: Functional dependence on the nervous system, hypothalamus
and pituitary Functional dependence on peripheral hormone carrier molecules
(e.g. corticosteroid binding globulin) Large number of potential toxicological targets such as enzymes
(both within the adrenal and systemically), receptors and biochemical functional mediators (e.g. adrenomedullin) - Glucocorticoid and mineralocorticoid production are also at the
end of pathways of sequentially-dependent steroidogenic steps and most vulnerable to upstream toxicity, e.g. by inhibition of any of the steroidogenic enzymes It is not possible based on current knowledge to rank the relative importance of each of the above attributes, however a relatively large literature has developed recording that the modulation or inhibition of steroidogenic enzymes is a major mechanism. This literature has extensively used the H295R cell
Fig. 1 illustrates the steroidogenic pathway in the three zones of the adrenal cortex (zona glomerulosa, zona fasciculata and zona reticularis). This figure also indicates an important difference between human and rat adrenocortical steroidogenesis in that rodents lack CYP17 (e.g. [7]) and therefore, cannot produce cortisol: this may limit their usefulness in detecting compounds that affect this key enzyme in steroidogenesis. An important point arising from this is that the steroidogenic capability of cells within a zone depends on their capability to express the various enzymes to metabolise precursor steroid substrates on to the next steroid in the pathway. The lack of CYP17 in that rat precludes the conversion of pregenenolone and progesterone to the 17-a forms, and hence production of 11-deoxycortisol and finally cortisol. As a result the rat pathway is via 11-deoxycorticosterone and the major glucocorticoid secreted is corticosterone. Corticosterone is the substrate for aldosterone production in both humans and rat. Also noted in Fig. 1 are the sites on action of classic adrenocortical enzyme inhibitors such as aminoglutethimide, etomidate, ketoconazole and cyanoketone. Harvey et al. [2,3,15] have reviewed the range of compounds known to affect adrenocortical function and collated over 70 examples of chemicals and their toxicological target within the adrenal. An abbreviated list is given in Table 1 from which it can be seen that every step in the steroid biosynthetic pathway, from the ACTH receptor and cholesterol transportation via steroid acute regulatory protein (StAR), to critical CYP and hydroxysteroid dehydrogenase enzymes in steroid synthesis are known targets for disruption. Of interest is that known adrenocortical endocrine disrupting compounds comprise a diverse group of chemical classes, and that a single compound can affect multiple adrenocortical targets. For example, aminoglutethimide down regulates adrenocortical ACTH receptors, is an inhibitor of CYP11A1 (cholesterol side chain cleavage) and CYP11B1 the terminal essential enzyme of glucocorticoid production, both cortisol and corticosterone. Polychlorinated biphenyls
Cholesterol + StAR CYP11A1
A
Pregnenolone
K
17α-Hydroxypregnenolone
K
HSD3B2
C
Dehydroepiandrosterone CYP17
CYP17 HSD3B2
C
C
HSD3B2
Progesterone
K
17α-Hydoxyprogesterone
K
11-Deoxycorticosterone CYP11B1 CYP11B2
AE
Corticosterone CYP11B2
K
CYP17
CYP17 CYP21
Androstenedione
CYP21
K
11-Deoxycortisol CYP11B1
AE
SULT2A1
DHEA-sulfate
ZONA RETICULARIS
Cortisol ZONA FASCICULATA
Aldosterone ZONA GLOMERULOSA
CYP17 is poorly expressed in rat and mouse consequently corticosterone is the dominant glucocorticoid produced
Multiple Target Steroidogenic Enzyme Inhibitors A= Aminoglutethimide C= Cyanoketone E= Etomidate K= Ketoconazole
Fig. 1. The human and rat steroidogenic pathways with sites of steroidogenic enzyme inhibition. Adapted from Harvey and Sutcliffe [5] with permission from John Wiley and Sons. See Table 1 and Refs. [2,3,15] for additional examples of chemicals inhibiting adrenocortical steroidogenic enzymes and references. Abbreviations: StAR, steroidogenic acute regulatory protein; CYP, cytochrome P450; HSD3B2, 3B-hydroxysteroid dehydrogenase type II; DHEA, dehydroepiandrosterone; SULT2A1, DHEA sulfotransferase.
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Table 1 Examples of chemicals inducing adrenocortical toxicity and targets in steroidogenic pathway. Target
Compound
Reference
ACTH receptor – Steroid acute regulatory (StAR) protein
Aminoglutethimidea
[16]
Econazole, miconazole, lindane Bromocriptinea Spironolactonea
[17–19] [20] [21]
Aminoglutethimidea Bromocripitinea
[22,23] [20]
Spironolactonea PCB126 Penta, octa, deca-brominated diphenyl ethers, tetrabromobisphenol-A Ketoconazolea
[24] [25] [26] [22]
Cyanoketone Trilostanea PCBs (101, 110, 126, 149) PAHs/PCBs Bromophenols, polybrominated biphenyls, 2,3,7,8-tetrabromodibenzo-p-dioxin, 2,3,7,8-terabromodibenzofuran Pioglitazonea
[27] [28] [29,30] [31]
PCBs (101, 110, 126, 149)
[29]
RU486a Ketoconazolea Flavonoids PCB126 PAHs/PCBs
[33] [22] [34] [25] [30]
Metyraponea Mitotane (o,p-DDD)a , MeSO2-DDE Etomidatea Ketoconazolea , aminoglutethimidea Flavonoids PCB126 PCBs (101, 110, 126, 149) Efonidipinea , mibefradil
[22,35] [22,36,37] [38,39] [22] [34] [25] [29] [40]
Prochloraz, imazalil Triazines, atrazine, simazine, propazine Di-, tributyl and phenyltin chlorides Fadrozolea PCBs (101, 110, 126, 149) Amoxicillina
[41] [42] [43] [44] [29] [45]
PCB126 Fadrozolea PCBs (101, 110, 126, 149) Efonidipinea , mibefradila PAHs/PCBs Amoxicillina , erythromycina
[25] [44] [29] [40] [30] [45]
– CYP11A1 (CYPscc) – CYP17
– 3-Hydroxysteroid dehydrogenase D4,5 isomerase
– 17b-Hydroxysteroid dehydrogenase – CYP21
– CYP11B1 (CYP11b/18)
– CYP19 (aromatase)
– CYP11B2 (aldosterone synthase)
a
[32]
Indicates pharmaceutical medicine (current or historical). The remaining examples are chemicals (industrial, agrochemicals, environmental pollutants, etc.).
(PCB’s) and related compounds collectively have a number of toxicological targets affecting CYP17, CYP21, CYP11B1, CYP11B2, CYP19 (aromatase) and the dehydrogenases 3-hydroxysteroid dehydrogenase D4,5 isomerase and 17b-hydroxysteroid dehydrogenase. These chemicals are environmental pollutants, are detectable in human tissues, and such findings raise the question of whether low level cumulative exposures could impair adrenocortical function in humans and wildlife alike. 4. Manifestation and evidence of adrenocortical toxicity 4.1. Human clinical experience The human drugs aminoglutethimide and etomidate are the classic examples of pharmaceutical medicines inducing fatal adrenocortical toxicity via off target unrecognised adrenocortical insufficiency resulting in Addisonian crisis, cardiovascular collapse
and death. The two drugs make interesting comparison based on the patient population and although aminoglutethimide has 3 sites of action (ACTH receptor down regulation, inhibition of CYP11A1 and CYP11B1) it was given as a sedative/anti-depressant to essentially healthy individuals and took longer for toxicity to express. Etomidate is an anaesthetic induction agent, by definition applied to patients undergoing various degrees of surgical trauma, and it was discovered quite quickly to induce fatal adrenocortical insufficiency (subsequently identified to be due to potent CYP11B1 inhibition) in surgical trauma patients that required a fully functional HPA axis [38] (see discussions in [15,39]). This adverse drug reaction may have remained hidden for longer except for the patient population who were unable to physiologically respond to the trauma and stress or of surgery and indeed their underlying medical condition. Continuous infusion of etomidate fell from favour because of the reports of adrenal crises but its use as a single bolus dose is still common and this too has been shown
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Apparently counter intuitively, increased adrenal weight may also be a sign of adrenal insufficiency. In this case the increase in gland size and weight, often confirmed by histopathological examination as thickening of the zona fasciculata, is invariably caused by a history of ACTH over stimulation of the gland. The finding of adrenocortical hypertrophy alone does not provide evidence of the mechanism. It has often been attributed to stress but in the absence of clear corroboratory evidence that the adrenal is functional such a conclusion cannot be supported. In stress, the adrenal cortex is functional and if the ACTH stimulation is sufficient to cause hypertrophy it is certainly sufficient to provoke an increase in glucocorticoid secretion. If cortisol or corticosterone is not measured then other markers of stress induced glucocorticoid secretion should be examined and the classic corroboratory stress related change is atrophy of the thymus with lymphocytolysis. If this is seen then this is at least some indication of adrenocortical competence arising from the expected concomitant stress-induced increase in glucocorticoid secretion. If this is not seen, and there is no other supportive stress related change, then stress as the mechanism of adrenocortical hypertrophy cannot be confirmed and further work is necessary to prove adrenocortical competence (see [5] and discussion later). Harvey and Sutcliffe [5] provide a full strategy for distinguishing stress from adrenocortical toxicity and steroidogenic inhibition and discuss the principles of adverse versus non-adverse findings. As is discussed below, the fact that the mechanism of adrenocortical steroidogenic inhibition is known does not mean it is non-adverse. The alternative cause of adrenocortical hypertrophy is a direct pharmacotoxicological effect on the steroidogenic pathway. In this case, an effect on adrenocortical steroidogenesis compromises glucocorticoid production/secretion and the reduction of circulatory glucocorticoid results in a loss of negative feedback inhibition at the hypothalamus and pituitary such that ACTH secretion is increased (see Fig. 2). This increased ACTH causes growth and hypertrophy of the adrenal. Drugs and chemicals are well documented to disrupt adrenocortical steroidogenesis at every stage in the pathway (see Table 1). Even acute, reversible “pharmacological” inhibition of glucocorticoid production can be
to result in significant adrenal insufficiency crises [46]. Etomidate is the case example of the consequences of unpredicted human adrenocortical toxicity resulting in markedly and fatally reduced cortisol and aldosterone secretion, and also demonstrates the importance of the adrenal and glucocorticoid action in quenching overwhelming inflammation considered to be the most important action of glucocorticoids in the stress response [14]. 4.2. Regulatory toxicology A defined set of regulatory toxicology studies must be conducted on all crop protection chemicals, pharmaceuticals and on certain chemicals that meet set criteria (e.g. human exposure potential, tonnage, uses, etc.). Typically these involve repeat dose studies (usually from 1 week to 1 year duration) in at least two standard laboratory species, usually the rat and dog, by the probable route of exposure which is usually oral. The measurement of hormones is not a regulatory requirement and thus the first indication of adrenal effects will be at necropsy and microscopic histopathology of the adrenal (the latter being a guideline requirement of all regulatory protocols). The first indication of an effect on the adrenal is a change in organ weight followed by histopathological correlates. Harvey and Sutcliffe [5] have provided extensive discussion and a rationale for distinguishing the mechanisms behind adrenal hypertrophy which is the most common finding. Changes in adrenal size and weight are invariably a result of the history of ACTH action on the gland. Adrenal atrophy commonly occurs with glucocorticoid agonists where their action is similar to endogenous steroids in providing negative feedback inhibition to hypothalamus and pituitary and reduction of ACTH secretion, resulting in a loss of trophic support to the gland. Adrenal atrophy may express as reduced size and weight but in the case of marked ACTH suppression it manifests as a marked thinning of the zona fasciculata upon microscopic examination ([13] and see discussions in [12,15]). Patients on long term glucocorticoid therapy lose the ability to maintain their own adequate glucocorticoid levels and upon withdrawal will experience adrenal insufficiency.
Stress
Normal
Toxic Inhibition
CNS Pathways Noxious Stimuli
Hypothalamus
Compensatory ACTH Secretion
CRH + AVP
Pituitary
ACTH
(-)
Normal Metabolic Functions
Hypothalamus
Hypothalamus
Pituitary
Pituitary
Glucocorticoid
Adrenal Cortex
Reduced (-) Feedback
Lymphoid atrophy Increased
Increased
ACTH
ACTH Increased
(+)
5
Glucocorticoid
Abolished or Decreased
Glucocorticoid
Adrenal Cortex
Adrenal Cortex
Hypertrophy
Hypertrophy
Stress Secretion of Glucocorticoid to Quench Inflammation
Direct Adrenal Toxicity Impaired Steroidogenesis Adrenal Insufficiency
Fig. 2. Pituitary–adrenocortical profiles: adrenal hypertrophy results from stress and adrenocortical steroidogenesis inhibition through the common mechanism of elevated ACTH. Adapted from Harvey and Sutcliffe [5] with permission from John Wiley and Sons. See Harvey and Sutcliffe [5] for full discussion and histopathological examples of stress changes in the adrenal cortex compared with toxicity. Abbreviations: ACTH, adrenocorticotrophic hormone; AVP, arginine vasopressin; CRH, corticotrophin-releasing hormone.
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a serious adverse effect to humans as the case of CYP11B1 inhibition by a single bolus dose of etomidate demonstrates ([46] and see discussions in [15,38,39]). An important point arising from Fig. 2 is that both stress and adrenocortical inhibition cause adrenocortical hypertrophy (increase in adrenal gland size/ weight or microscopic hypertrophy of cells) and it is not possible to make a conclusion on mechanism with this finding alone. This is because both are associated with increased secretion of ACTH. It is the reason for the increase in ACTH which is critical. The increased ACTH secretion in the stress response is due to central perception of noxious stimuli activating neural and neuroendocrine pathways whilst adrenocortical steroidogenic toxicity results in loss of negative feedback control of ACTH. In stress the adrenal cortex remains functional and capable of producing glucocorticoids, whilst in steroidogenic pathway inhibition it is impaired and this forms the basis for distinguishing mechanisms (in addition to the other evidence that may assist a differential diagnosis such as the plausibility of stress including adverse clinical signs and surrogate markers of elevated glucocorticoid action in the body such as atrophy of the thymus as previously mentioned). However, a useful indicator of adrenocortical toxicity versus stress is dependent on the magnitude of the adrenal hypertrophy, in turn dependent on the duration and magnitude of ACTH stimulation, and potent adrenocortical enzyme inhibitors resulting in sustained unopposed ACTH secretion, such as cyanoketone, can cause a 100% increase in adrenal weights in 3 days [47] a magnitude rarely seen with stress. Continuous ACTH infusion, mimicking the state of complete adrenocortical glucocorticoid inhibition and unopposed ACTH secretion, has been reported to result in a 10-fold increase in adrenal weight in 18 days [5,48]. Mechanistic models of assessing adrenocortical function, both in vivo models exploiting the action of ACTH in adrenocortical function, and in vitro systems for identifying specific sites and targets of toxicity, are discussed later. 4.3. Wildlife The manifestation of adrenocortical toxicity in wildlife is more difficult to recognise in the field but follows the same principles as laboratory animals. It is now becoming apparent that birds [49], fish [8] and marine mammals [9] are showing evidence of impaired adrenocortical function, ill health and reduced survival fitness linked to environmental chemical exposures. Recently, a population of dolphins exposed to the Deep Water Horizon oil spill pollution have been shown to have impaired cortisol and aldosterone secretion [9] compared with populations in nonpolluted areas. Aquatic species are often considered sentinel species for other wildlife but also the general health of the environment in which humans may also be exposed directly or indirectly. Chemical residues entering the food chain are well known to accumulate and form secondary exposure routes. Impairment of adrenocortical function at the population level may be expected to reduce survival fitness and may simply manifest in non-specific population declines. At the individual level the inability to adapt physiologically to adverse environmental insults may contribute to increased vulnerability, morbidity and mortality. The multitude of chemicals known to affect adrenocortical function via different target sites in the steroidogenic pathway may also act in concert: cumulative low level exposures may be as mixtures, or sequential exposures to different agents, increasing risk of adverse outcomes. There is a literature gap on the potential effects of low level mixtures and the dynamic physiological nature of the HPA axis raises the possibility that chemical exposures may result in occult subclinical blunting of the stress response, rather than frank abolition of glucocorticoid production, which would nevertheless impair survival fitness.
5. Models of adrenocortical assessment 5.1. In vivo approaches to adrenocortical functional evaluation As mentioned previously it is not possible to distinguish a mechanism from the observation of adrenocortical hypertrophy alone but it is important to prove functionality of the adrenal cortex when this finding is seen and to distinguish stress from adrenal toxicity (stress should result in an elevated glucocorticoid response to elevated endogenous ACTH and not result in impaired adrenocortical function). A simple test is to conduct an ACTH challenge study where injection of ACTH should provoke the secretion of glucocorticoid: the concept is in proving functionality of the adrenal cortex. As adrenocortical toxicants/enzyme inhibitors impair steroidogenesis, ACTH challenge in the species showing the effect will produce a blunted or lower response compared with controls. Potent adrenocortical enzyme inhibitors can produce marked inhibition of steroidogenesis after single or a few repeat exposures (e.g. etomidate [46] and cyanoketone [47]) and thus, the study can be relatively short duration. Designs can include positive controls but the basic protocol is to administer the test drug at a dose known to result in the adrenal effect and after a defined number of days administer ACTH, take blood samples at defined time points thereafter and measure the adrenocortical response, corticosterone levels in rats or mice and cortisol in dogs etc. This is a routine diagnostic test in human and veterinary clinical endocrinology to diagnose adrenocortical insufficiency conditions such as Addison’s disease and its variants. This test has been previously proposed to have utility in regulatory toxicology and adrenal endocrine disruption research [2,3,5,12,15]. By measuring additional steroid profiles it may also be possible to establish where in the pathway the drug/chemical may be exerting its effect. For example, if progesterone secretion is also reduced, it may be inferred that the inhibition is upstream and may therefore be 3-hydroxysteroid dehydrogenase D4,5 isomerase, CYP11A1 (cholesterol side chain cleavage), StAR or even down regulation of the ACTH receptor itself (shown to occur with aminoglutethimide [16]). However, the best tools for examining molecular sites of toxicity are cell lines. 5.2. In vitro adrenocortical model: H295R cell line Currently, the best available in vitro cell based model for studying adrenocortical function is the H295R cell line [3,15,50,51]. The H295R cell line is derived from a human adrenocortical carcinoma considered to arise from the border of the zona glomerulosa and zona fasciculata since it has the capability to produce both aldosterone and cortisol. Indeed it has virtually universal steroidogenic capability and thus it can be used to assess the entire process of steroidogenesis and it is now a validated test for androgen production in the USEPA battery of endocrine disruptor screening tests. This cell line does not express the ACTH receptor as well as normal cells specifically derived from the zona fasciculata, probably due to the phenotypic influence of its zona glomerulosa origin, and thus ACTH challenge is less effective and forskolin is used [51] which mimics the action of ACTH on signal transduction via cAMP. A typical protocol is to culture the cells in the presence of a number of test concentrations of the drug or chemical, challenge with forskolin and measure the resultant steroid secretion, aldosterone or cortisol. Evaluating steroid profiles (e.g. [40,44]) can provide information on the site of toxicity, but specific enzymes [30,43–45] and gene expression [19,32,45] can be directly measured. Fuller discussions of the utility of in vitro models and specifically the utility of the H295R cell line can be found in [15,50,51]. However, Harvey et al. [2,3,15] have suggested that this would be useful to develop and validate
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for the assessment of cortisol and aldosterone secretion which is currently lacking in any regulatory endocrine disruption strategy. Indeed, the H295R cell line continues to be enhanced. The use of gas chromatography tandem mass spectrometry analysis of H295R media has been developed, and used to evaluate steroid profiles, and compared with metabolomics methodology using ultra performance liquid chromatography–time-of-flight–mass-spectrometry (UPLC–ToF–MS) to identify specific “fingerprints” of endocrine disrupting chemicals (see [52,53]). It has also been proposed to use this cell line, along with other assays, in an integrated testing strategy for oestrogenicity to broaden sensitivity in identification of endocrine disruption through this mechanism [54]. Although the endpoints reported in these studies [52–54] pertain largely to sex steroids, they can also be applied to develop fingerprints using cortisol, aldosterone and other steroids specific to adrenocortical function. Such a development to the OECD Test Guideline on H295R in endocrine disruption evaluation [55], which currently only considers production of 17b-oestradiol and testosterone as endpoints, and validation of cortisol and aldosterone as endpoints, would be a major step in recognizing the unique role of the adrenal cortex and the extent to which glucocorticoid and/or mineralocorticoid producing adrenocortical cells may be a target for endocrine disruption. 6. Conclusion Evidence suggests that the adrenal cortex is a common toxicological target for both drugs and chemicals and inhibition of steroidogenic pathways is considered the major mechanism of action. The range of chemicals shown to inhibit adrenocortical steroidogenesis is diverse and there are multiple examples demonstrating that every step and enzyme in mineralocorticoid and glucocorticoid pathways can be affected. Human adrenocortical conditions, such as adrenogenital and salt wasting syndromes in infants, have defined molecular aetiologies related to problems with adrenocortical steroidogenic enzyme deficiencies [56–58] raising the possibility that environmental chemical exposure may provide a risk factor in promoting phenotypic variants of the disease at variable severities. The physiological role of the adrenal cortex can also hide expression of toxicity which may only become apparent when significant activation of the stress response is required to tolerate trauma and crises and assist survival. The lack of standardised adrenocortical functional testing is considered a major deficiency in regulatory endocrine disrupter strategies (see [2,3,59]) and more surprising given that the adrenal is the most common toxicological target in the endocrine system [4] its role as a vital organ and in developmental processes. Further, the tendency to “write-off” adrenal findings as stress in mammalian regulatory toxicology studies, in the absence of corroboratory evidence of adrenocortical functionality, is common and potentially dangerous which requires recognition in regulatory toxicology. Of most importance is that stress is not the only cause of adrenal hypertrophy in vivo: partial adrenocortical steroidogenic inhibition may co-exist with general toxicity induced stress, or operate independently, with expression being a function of relative drug/ chemical potency, dose and duration of exposure. It has been suggested that all cases of adrenocortical hypertrophy warrant further mechanistic investigation [5] and that a plausible supportive case based on additional evidence of adrenocortical competency should be made for all claims of a stress mechanism. The measurement of glucocorticoids is diagnostic, and the distinguishing feature between adrenocortical hypertrophy induced by stress versus steroidogenic toxicity is the adequacy of the glucocorticoid response. This can be assessed in vivo in the ACTH challenge study or in vitro in the H295R cell line. Finally, although
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controlled by the renin-angiotensin system rather than ACTH, the competency of production of aldosterone is also considered of major importance and it is suggested that this is also assessed in all cases of drugs or chemicals causing adrenocortical hypertrophy.
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[44] U. Muller-Vieira, M. Angotti, R.W. Hartman, The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11 B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors, J. Steroid Biochem. Mol. Biol. 96 (2005) 259–270. [45] T. Gracia, K. Hilscherova, P.D. Jones, et al., Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds, Toxicol. Appl. Pharmacol. 225 (2007) 142–153. [46] J.B. Lundy, M.L. Slane, J.D. Frizzi, Acute adrenal insufficiency after a single dose of etomidate, J. Intensive Care Med. 22 (2007) 111–117. [47] S.F. Akana, J. Shinsako, M.F. Dallman, Drug-induced adrenal hypertrophy provides evidence for reset in the adrenocortical system, Endocrinology 113 (1983) 2232–2237. [48] J. Pudney, G.M. Price, B.J. Whitehouse, G.P. Vinson, Effects of chronic ACTH stimulation on the morphology of the rat adrenal cortex, Anat. Rec. 210 (1984) 603–615. [49] R. Baos, J. Blas, Adrenal toxicology in birds: environmental contaminants and the avian response to stress, in: P.W. Harvey, D.J. Everett, C.J. Springall (Eds.), Adrenal Toxicology, Informa Healthcare, New York, 2009, pp. 257–293. [50] T. Sanderson, Adrenocortical toxicology in vitro: assessment of steroidogenic enzyme expression and steroid production in H295R cells, in: P.W. Harvey, D.J. Everett, C.J. Springall (Eds.), Adrenal Toxicology, Informa Healthcare, New York, 2009, pp. 175–182. [51] J. Parmar, W. Rainey, Comparisons of adrenocortical cell lines as in vitro test systems, in: P.W. Harvey, D.J. Everett, C.J. Springall (Eds.), Adrenal Toxicology, Informa Healthcare, New York, 2009, pp. 183–204. [52] J.C.W. Rijk, A.A.C.M. Peijnenburg, M.H. Blokland, A. Lommen, L.A.P. Hoogenboom, T.F.H. Bovee, Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach, Chem. Res. Toxicol. 25 (2012) 1720–1731. [53] M. Reitsma, T.F.H. Bovee, A.A.C.M. Peijnenburg, P.J. Hendriksen, L.A.P. Hoogenboom, J.C.W. Rijk, Endocrine disrupting effects of thioxanthone photoinitiators, Toxicol. Sci. 132 (2013) 64–74. [54] S. Wang, J.C. Rijk, H.T. Besselink, R. Houtman, A.A. Peijnenburg, A. Brouwer, I.M. Rietjens, T.F. Bovee, Extending an in vitro panel for estrogenicity testing: the added value of bioassays for measuring antiandrogenic activities and effects on steroidogenesis, Toxicol. Sci. (2014), doi:http://dx.doi.org/10.1093/toxsci/ kfu103. [55] OECD, Test No. 456: H295R steroidogenesis assay, OECD Guidelines for the Testing of Chemicals. Available at: http://www.oecd-ilibrary.org/environment/test-no-456-h295r-steroidogenesis-assay_9789264122642-en. [56] S.R. Bornstein, Predisposing factors for adrenal insufficiency, N. Engl. J. Med. 360 (2009) 2328–2339. [57] T. Hakki, R. Bernhardt, CYP17- and CYP11B-dependent steroid hydroxylases as drug development targets, Pharmacol. Ther. 111 (2006) 27–52. [58] R.I.G. Holt, N.A. Hanley, An overview of human adrenal dysfunction, in: P.W. Harvey, D.J. Everett, C.J. Springall (Eds.), Adrenal Toxicology, Informa Healthcare, New York, 2009, pp. 39–75. [59] P.W. Harvey, D.J. Everett, Regulation of endocrine-disrupting chemicals: critical overview and deficiencies in toxicology and risk assessment for human health, Best Pract. Res. Clin. Endocrinol. Metab. 20 (2006) 145–165.
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Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
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Review
Adrenocortical endocrine disruption Philip W. Harvey * Toxicology Department, Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2014 Received in revised form 12 September 2014 Accepted 14 October 2014 Available online xxx
The adrenal has been neglected in endocrine disruption regulatory testing strategy. The adrenal is a vital organ, adrenocortical insufficiency is recognised in life threatening “adrenal crises” and Addison’s disease, and the consequences of off-target toxicological inhibition of adrenocortical steroidogenesis is well recognised in clinical medicine, where drugs such as aminoglutethimide and etomidate killed patients via unrecognised inhibition of adrenocortical steroidogenic enzymes (e.g. CYP11B1) along the cortisol and aldosterone pathways. The consequences of adrenocortical dysfunction during early development are also recognised in the congenital salt wasting and adrenogenital syndromes presenting neonatally, yet despite a remit to focus on developmental and reproductive toxicity mechanisms of endocrine disruption by many regulatory agencies (USEPA EDSTAC; REACH) the assessment of adrenocortical function has largely been ignored. Further, every step in the adrenocortical steroidogenic pathway (ACTH receptor, StAR, CYP’s 11A1, 17, 21, 11B1, 11B2, and 3-hydroxysteroid dehydrogenase D4,5 isomerase) is known to be a potential target with multiple examples of chemicals inhibiting these targets. Many of these chemicals have been detected in human and wildlife tissues. This raises the question of whether exposure to low level environmental chemicals may be affecting adrenocortical function. This review examines the omission of adrenocortical testing in the current regulatory frameworks; the characteristics that make the adrenal cortex particularly vulnerable to toxic insult; chemicals and their toxicological targets within the adrenocortical steroidogenic pathways; the typical manifestations of adrenocortical toxicity (e.g. human iatrogenically induced pharmacotoxicological adrenal insufficiency, manifestations in typical mammalian regulatory general toxicology studies, manifestations in wildlife) and models of adrenocortical functional assessment. The utility of the in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled ‘Endocrine disruptors and steroids’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Adrenal cortex Adrenocortical insufficiency Adrenal toxicity Adrenal hypertrophy H295R cell line Steroidogenesis Stress Toxicity
Contents 1. 2. 3. 4.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of the adrenal gland conveying vulnerability to toxicity . . . . . . . . . . . . . . . . Chemicals and toxicological targets within the steroidogenic pathway of the adrenal cortex Manifestation and evidence of adrenocortical toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human clinical experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Regulatory toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Wildlife . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Models of adrenocortical assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo approaches to adrenocortical functional evaluation . . . . . . . . . . . . . . . . . . . . . 5.1.
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* Tel.: +44 1423848421. E-mail address: [email protected] (P.W. Harvey). http://dx.doi.org/10.1016/j.jsbmb.2014.10.009 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
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5.2. In vitro adrenocortical model: H295R cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction The main focus of endocrine disruption interest has centred on reproductive and developmental endpoints, largely dictated by a regulatory framework for the testing and registration of chemicals arising from the United States Environmental Protection Agencies (USEPA) Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations for specific studies enforced in the mid 1990s [1] and later adopted by other regulatory initiatives. Specific evaluation of compounds for (anti) estrogenic and (anti-) androgenic properties in a range of in vitro and in vivo studies, in a tiered approach triggered by findings, forms the core of the testing requirements and the protocols are followed by regulatory toxicology registrants and the research community alike, to a greater or lesser degree of robustness. Recognition that some developmental processes, particularly in wildlife species, are controlled by thyroid function led to the incorporation of some tests to examine this gland. In terms of an overall strategy for endocrine disruption this regulatory framework is lacking because it only focuses on reproductive and developmental endpoints and ignores the integrated function of the endocrine system as a whole and particularly the adrenal gland [2,3] although for chemical registrations there will usually be a range of chronic toxicity and carcinogenicity studies with full microscopic histopathological evaluation of all organs and tissues including the endocrine system (pituitary, thyroid, parathyroid, pancreas, adrenal, ovary and testis) and endocrine target tissues (e.g. mammary, uterus, prostate, etc.). The problem arises in how to interpret toxicopathological changes in these non reproductive endocrine organs, particularly the adrenal as the most common toxicological target in the endocrine system [4], without additional supportive mechanistic studies. A particular weakness has centred on the interpretation of the most commonly observed change in the adrenal namely adrenal (adrenocortical) hypertrophy. Adrenal hypertrophy (increase in gland weight or increase in zone width or cell size microscopically) is often dismissed as stress in the absence of any corroboratory evidence of retained adrenocortical functionality, which is ignorant of the possibility of toxicity in the steroidogenic pathway resulting in glucocorticoid insufficiency. In such cases, glucocorticoid insufficiency results in the loss of feedback regulation to hypothalamus and pituitary and in compensatory over secretion of adrenocorticotrophic hormone (ACTH) and this stimulation promotes growth/ hypertrophy in the zona fasciculata and visibly larger and heavier adrenals [5]. Whilst the current EDSTAC related screening strategies have ignored the adrenal (surprisingly given its role as a vital organ and involvement in development processes, where human conditions such as congenital adrenogenital syndrome or salt wasting conditions expressing neonatally are evidence of the consequences of developmental adrenocortical dysfunction) its importance has been recently recognised by the United States Food and Drug Administration (USFDA) guidance on Endocrine Disruption Potential of Drugs: Non-Clinical Guidance [6]. These draft recommendations recognise the need for enzyme assays including CYP11A1, 11B1, 11B2, 17A1 and 21A1 which encompass the steroidogenic pathways relevant for adrenocortical glucocorticoid and mineralocorticoid metabolism. These recommendations also suggest that endocrine function studies (e.g. measuring hormone levels) are employed to elucidate mechanisms of
histopathological changes seen in endocrine organs specifically including the adrenal, and recognise the adrenal/glucocorticoid action in early life developmental processes (e.g. bone growth). Essentially, these draft guidelines propose the need for full supportive endocrine evaluation when indicated by any changes (usually microscopic histopathology in the non clinical study protocols used) detected in the endocrine system and delineate approaches for expected changes (e.g. drug pharmacological action) from the unexpected. Indeed, the experience of the unexpected adrenocortical toxicity of drugs reaching the market such as aminoglutethimide and etomidate [2,3,5,7] has demonstrated that off target adrenocortical steroidogenic enzyme inhibition can produce fatal adrenal insufficiency in patients. In turn, because of commonality of mechanisms, this raises the issue of whether low level environmental chemical exposures may be impairing adrenocortical function in humans and wildlife. Aquatic wildlife are often considered sentinel species (they are continuously exposed to chemicals in water) and evidence is emerging of adrenocortical endocrine disruption in fish [8] and dolphins [9] as measured by impaired cortisol production, and in the dolphin study impaired aldosterone production was also reported. This brief review will now outline the properties of the adrenal cortex that impart vulnerability to toxic insult, the range of chemicals known to affect the adrenal and their targets within the steroidogenic pathway, models of adrenocortical assessment including species limitations and in vitro test systems (H295R adrenocortical cell line), and approaches to distinguishing stress related changes from toxicity when the common finding of adrenal hypertrophy is observed. Full reviews of adrenal toxicity and toxicologic pathology can be found elsewhere [10–13]. 2. Characteristics of the adrenal gland conveying vulnerability to toxicity The adrenal gland is the most commonly affected endocrine organ in toxicology studies [4]. Part of the reason for this is the central physiological role of the adrenal in the stress response, where toxic insult from the administration of drugs and chemicals administered at the maximum tolerated dose is often stressful and activates the hypothalamo–pituitary–adrenocortical (HPA) axis to increase ACTH secretion. The characteristic changes consistent with increased ACTH stimulation of the adrenal gland are increased size and weight macroscopicallyandincrease in zona fasciculata thicknessand/orcell size microscopically, comprising the typical actions after which this trophic hormone was named. The mechanism of HPA activation and the stress response to environmental insults, including toxicity, is a survival mechanism designed to modify the animals ability to cope physiologically and metabolically to adverse conditions primarily effected by increased glucocorticoid exposure and whilst glucocorticoids have a number of actions (e.g. glucose utilisation and diversion of metabolic resources) it is considered that actions of these steroids (cortisol or corticosterone depending on species) in quenching inflammation which could otherwise overwhelm the organism is the vital action of this system [14]. Indeed, it is the same physiological and anatomical attributes of the HPA axis that are required to effectively modulate the stress response (e.g. rich blood supply to rapidly distribute steroids systemically) that also convey vulnerability of the adrenal to exposure to toxicants and in turn toxic insult. Such properties have been identified [7,12] and are listed below:
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High vascularity - Disproportionately large blood volume received per unit mass of
3
line (discussed later) and documents that almost every step in adrenocortical steroidogenesis is known to be disrupted by a chemical and this is discussed in Section 3.
adrenal tissue ensuring exposures to toxicants Lipophilicity due to rich cholesterol and steroid content
3. Chemicals and toxicological targets within the steroidogenic pathway of the adrenal cortex
favouring local deposition of lipophilic compounds High content of unsaturated fatty acids in adrenocortical cell
membranes susceptible to damage by lipid peroxidation both directly (via parent compound or its metabolites) and indirectly (via generation of reactive species such as free radicals) High content of cytochrome P450 (CYP) enzymes present in the adrenal cortex which can produce: - Reactive parent compound metabolites of toxicants that then
mediate toxicity - Hydroxylation reactions that may generate free radicals which
then damage adrenocortical cells and membrane (as above) In addition, the adrenal cortex is the final effector organ in a chain of events and factors elsewhere in the HPA system, and peripherally, may influence adrenocortical function, such as: Functional dependence on the nervous system, hypothalamus
and pituitary Functional dependence on peripheral hormone carrier molecules
(e.g. corticosteroid binding globulin) Large number of potential toxicological targets such as enzymes
(both within the adrenal and systemically), receptors and biochemical functional mediators (e.g. adrenomedullin) - Glucocorticoid and mineralocorticoid production are also at the
end of pathways of sequentially-dependent steroidogenic steps and most vulnerable to upstream toxicity, e.g. by inhibition of any of the steroidogenic enzymes It is not possible based on current knowledge to rank the relative importance of each of the above attributes, however a relatively large literature has developed recording that the modulation or inhibition of steroidogenic enzymes is a major mechanism. This literature has extensively used the H295R cell
Fig. 1 illustrates the steroidogenic pathway in the three zones of the adrenal cortex (zona glomerulosa, zona fasciculata and zona reticularis). This figure also indicates an important difference between human and rat adrenocortical steroidogenesis in that rodents lack CYP17 (e.g. [7]) and therefore, cannot produce cortisol: this may limit their usefulness in detecting compounds that affect this key enzyme in steroidogenesis. An important point arising from this is that the steroidogenic capability of cells within a zone depends on their capability to express the various enzymes to metabolise precursor steroid substrates on to the next steroid in the pathway. The lack of CYP17 in that rat precludes the conversion of pregenenolone and progesterone to the 17-a forms, and hence production of 11-deoxycortisol and finally cortisol. As a result the rat pathway is via 11-deoxycorticosterone and the major glucocorticoid secreted is corticosterone. Corticosterone is the substrate for aldosterone production in both humans and rat. Also noted in Fig. 1 are the sites on action of classic adrenocortical enzyme inhibitors such as aminoglutethimide, etomidate, ketoconazole and cyanoketone. Harvey et al. [2,3,15] have reviewed the range of compounds known to affect adrenocortical function and collated over 70 examples of chemicals and their toxicological target within the adrenal. An abbreviated list is given in Table 1 from which it can be seen that every step in the steroid biosynthetic pathway, from the ACTH receptor and cholesterol transportation via steroid acute regulatory protein (StAR), to critical CYP and hydroxysteroid dehydrogenase enzymes in steroid synthesis are known targets for disruption. Of interest is that known adrenocortical endocrine disrupting compounds comprise a diverse group of chemical classes, and that a single compound can affect multiple adrenocortical targets. For example, aminoglutethimide down regulates adrenocortical ACTH receptors, is an inhibitor of CYP11A1 (cholesterol side chain cleavage) and CYP11B1 the terminal essential enzyme of glucocorticoid production, both cortisol and corticosterone. Polychlorinated biphenyls
Cholesterol + StAR CYP11A1
A
Pregnenolone
K
17α-Hydroxypregnenolone
K
HSD3B2
C
Dehydroepiandrosterone CYP17
CYP17 HSD3B2
C
C
HSD3B2
Progesterone
K
17α-Hydoxyprogesterone
K
11-Deoxycorticosterone CYP11B1 CYP11B2
AE
Corticosterone CYP11B2
K
CYP17
CYP17 CYP21
Androstenedione
CYP21
K
11-Deoxycortisol CYP11B1
AE
SULT2A1
DHEA-sulfate
ZONA RETICULARIS
Cortisol ZONA FASCICULATA
Aldosterone ZONA GLOMERULOSA
CYP17 is poorly expressed in rat and mouse consequently corticosterone is the dominant glucocorticoid produced
Multiple Target Steroidogenic Enzyme Inhibitors A= Aminoglutethimide C= Cyanoketone E= Etomidate K= Ketoconazole
Fig. 1. The human and rat steroidogenic pathways with sites of steroidogenic enzyme inhibition. Adapted from Harvey and Sutcliffe [5] with permission from John Wiley and Sons. See Table 1 and Refs. [2,3,15] for additional examples of chemicals inhibiting adrenocortical steroidogenic enzymes and references. Abbreviations: StAR, steroidogenic acute regulatory protein; CYP, cytochrome P450; HSD3B2, 3B-hydroxysteroid dehydrogenase type II; DHEA, dehydroepiandrosterone; SULT2A1, DHEA sulfotransferase.
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Table 1 Examples of chemicals inducing adrenocortical toxicity and targets in steroidogenic pathway. Target
Compound
Reference
ACTH receptor – Steroid acute regulatory (StAR) protein
Aminoglutethimidea
[16]
Econazole, miconazole, lindane Bromocriptinea Spironolactonea
[17–19] [20] [21]
Aminoglutethimidea Bromocripitinea
[22,23] [20]
Spironolactonea PCB126 Penta, octa, deca-brominated diphenyl ethers, tetrabromobisphenol-A Ketoconazolea
[24] [25] [26] [22]
Cyanoketone Trilostanea PCBs (101, 110, 126, 149) PAHs/PCBs Bromophenols, polybrominated biphenyls, 2,3,7,8-tetrabromodibenzo-p-dioxin, 2,3,7,8-terabromodibenzofuran Pioglitazonea
[27] [28] [29,30] [31]
PCBs (101, 110, 126, 149)
[29]
RU486a Ketoconazolea Flavonoids PCB126 PAHs/PCBs
[33] [22] [34] [25] [30]
Metyraponea Mitotane (o,p-DDD)a , MeSO2-DDE Etomidatea Ketoconazolea , aminoglutethimidea Flavonoids PCB126 PCBs (101, 110, 126, 149) Efonidipinea , mibefradil
[22,35] [22,36,37] [38,39] [22] [34] [25] [29] [40]
Prochloraz, imazalil Triazines, atrazine, simazine, propazine Di-, tributyl and phenyltin chlorides Fadrozolea PCBs (101, 110, 126, 149) Amoxicillina
[41] [42] [43] [44] [29] [45]
PCB126 Fadrozolea PCBs (101, 110, 126, 149) Efonidipinea , mibefradila PAHs/PCBs Amoxicillina , erythromycina
[25] [44] [29] [40] [30] [45]
– CYP11A1 (CYPscc) – CYP17
– 3-Hydroxysteroid dehydrogenase D4,5 isomerase
– 17b-Hydroxysteroid dehydrogenase – CYP21
– CYP11B1 (CYP11b/18)
– CYP19 (aromatase)
– CYP11B2 (aldosterone synthase)
a
[32]
Indicates pharmaceutical medicine (current or historical). The remaining examples are chemicals (industrial, agrochemicals, environmental pollutants, etc.).
(PCB’s) and related compounds collectively have a number of toxicological targets affecting CYP17, CYP21, CYP11B1, CYP11B2, CYP19 (aromatase) and the dehydrogenases 3-hydroxysteroid dehydrogenase D4,5 isomerase and 17b-hydroxysteroid dehydrogenase. These chemicals are environmental pollutants, are detectable in human tissues, and such findings raise the question of whether low level cumulative exposures could impair adrenocortical function in humans and wildlife alike. 4. Manifestation and evidence of adrenocortical toxicity 4.1. Human clinical experience The human drugs aminoglutethimide and etomidate are the classic examples of pharmaceutical medicines inducing fatal adrenocortical toxicity via off target unrecognised adrenocortical insufficiency resulting in Addisonian crisis, cardiovascular collapse
and death. The two drugs make interesting comparison based on the patient population and although aminoglutethimide has 3 sites of action (ACTH receptor down regulation, inhibition of CYP11A1 and CYP11B1) it was given as a sedative/anti-depressant to essentially healthy individuals and took longer for toxicity to express. Etomidate is an anaesthetic induction agent, by definition applied to patients undergoing various degrees of surgical trauma, and it was discovered quite quickly to induce fatal adrenocortical insufficiency (subsequently identified to be due to potent CYP11B1 inhibition) in surgical trauma patients that required a fully functional HPA axis [38] (see discussions in [15,39]). This adverse drug reaction may have remained hidden for longer except for the patient population who were unable to physiologically respond to the trauma and stress or of surgery and indeed their underlying medical condition. Continuous infusion of etomidate fell from favour because of the reports of adrenal crises but its use as a single bolus dose is still common and this too has been shown
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Apparently counter intuitively, increased adrenal weight may also be a sign of adrenal insufficiency. In this case the increase in gland size and weight, often confirmed by histopathological examination as thickening of the zona fasciculata, is invariably caused by a history of ACTH over stimulation of the gland. The finding of adrenocortical hypertrophy alone does not provide evidence of the mechanism. It has often been attributed to stress but in the absence of clear corroboratory evidence that the adrenal is functional such a conclusion cannot be supported. In stress, the adrenal cortex is functional and if the ACTH stimulation is sufficient to cause hypertrophy it is certainly sufficient to provoke an increase in glucocorticoid secretion. If cortisol or corticosterone is not measured then other markers of stress induced glucocorticoid secretion should be examined and the classic corroboratory stress related change is atrophy of the thymus with lymphocytolysis. If this is seen then this is at least some indication of adrenocortical competence arising from the expected concomitant stress-induced increase in glucocorticoid secretion. If this is not seen, and there is no other supportive stress related change, then stress as the mechanism of adrenocortical hypertrophy cannot be confirmed and further work is necessary to prove adrenocortical competence (see [5] and discussion later). Harvey and Sutcliffe [5] provide a full strategy for distinguishing stress from adrenocortical toxicity and steroidogenic inhibition and discuss the principles of adverse versus non-adverse findings. As is discussed below, the fact that the mechanism of adrenocortical steroidogenic inhibition is known does not mean it is non-adverse. The alternative cause of adrenocortical hypertrophy is a direct pharmacotoxicological effect on the steroidogenic pathway. In this case, an effect on adrenocortical steroidogenesis compromises glucocorticoid production/secretion and the reduction of circulatory glucocorticoid results in a loss of negative feedback inhibition at the hypothalamus and pituitary such that ACTH secretion is increased (see Fig. 2). This increased ACTH causes growth and hypertrophy of the adrenal. Drugs and chemicals are well documented to disrupt adrenocortical steroidogenesis at every stage in the pathway (see Table 1). Even acute, reversible “pharmacological” inhibition of glucocorticoid production can be
to result in significant adrenal insufficiency crises [46]. Etomidate is the case example of the consequences of unpredicted human adrenocortical toxicity resulting in markedly and fatally reduced cortisol and aldosterone secretion, and also demonstrates the importance of the adrenal and glucocorticoid action in quenching overwhelming inflammation considered to be the most important action of glucocorticoids in the stress response [14]. 4.2. Regulatory toxicology A defined set of regulatory toxicology studies must be conducted on all crop protection chemicals, pharmaceuticals and on certain chemicals that meet set criteria (e.g. human exposure potential, tonnage, uses, etc.). Typically these involve repeat dose studies (usually from 1 week to 1 year duration) in at least two standard laboratory species, usually the rat and dog, by the probable route of exposure which is usually oral. The measurement of hormones is not a regulatory requirement and thus the first indication of adrenal effects will be at necropsy and microscopic histopathology of the adrenal (the latter being a guideline requirement of all regulatory protocols). The first indication of an effect on the adrenal is a change in organ weight followed by histopathological correlates. Harvey and Sutcliffe [5] have provided extensive discussion and a rationale for distinguishing the mechanisms behind adrenal hypertrophy which is the most common finding. Changes in adrenal size and weight are invariably a result of the history of ACTH action on the gland. Adrenal atrophy commonly occurs with glucocorticoid agonists where their action is similar to endogenous steroids in providing negative feedback inhibition to hypothalamus and pituitary and reduction of ACTH secretion, resulting in a loss of trophic support to the gland. Adrenal atrophy may express as reduced size and weight but in the case of marked ACTH suppression it manifests as a marked thinning of the zona fasciculata upon microscopic examination ([13] and see discussions in [12,15]). Patients on long term glucocorticoid therapy lose the ability to maintain their own adequate glucocorticoid levels and upon withdrawal will experience adrenal insufficiency.
Stress
Normal
Toxic Inhibition
CNS Pathways Noxious Stimuli
Hypothalamus
Compensatory ACTH Secretion
CRH + AVP
Pituitary
ACTH
(-)
Normal Metabolic Functions
Hypothalamus
Hypothalamus
Pituitary
Pituitary
Glucocorticoid
Adrenal Cortex
Reduced (-) Feedback
Lymphoid atrophy Increased
Increased
ACTH
ACTH Increased
(+)
5
Glucocorticoid
Abolished or Decreased
Glucocorticoid
Adrenal Cortex
Adrenal Cortex
Hypertrophy
Hypertrophy
Stress Secretion of Glucocorticoid to Quench Inflammation
Direct Adrenal Toxicity Impaired Steroidogenesis Adrenal Insufficiency
Fig. 2. Pituitary–adrenocortical profiles: adrenal hypertrophy results from stress and adrenocortical steroidogenesis inhibition through the common mechanism of elevated ACTH. Adapted from Harvey and Sutcliffe [5] with permission from John Wiley and Sons. See Harvey and Sutcliffe [5] for full discussion and histopathological examples of stress changes in the adrenal cortex compared with toxicity. Abbreviations: ACTH, adrenocorticotrophic hormone; AVP, arginine vasopressin; CRH, corticotrophin-releasing hormone.
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a serious adverse effect to humans as the case of CYP11B1 inhibition by a single bolus dose of etomidate demonstrates ([46] and see discussions in [15,38,39]). An important point arising from Fig. 2 is that both stress and adrenocortical inhibition cause adrenocortical hypertrophy (increase in adrenal gland size/ weight or microscopic hypertrophy of cells) and it is not possible to make a conclusion on mechanism with this finding alone. This is because both are associated with increased secretion of ACTH. It is the reason for the increase in ACTH which is critical. The increased ACTH secretion in the stress response is due to central perception of noxious stimuli activating neural and neuroendocrine pathways whilst adrenocortical steroidogenic toxicity results in loss of negative feedback control of ACTH. In stress the adrenal cortex remains functional and capable of producing glucocorticoids, whilst in steroidogenic pathway inhibition it is impaired and this forms the basis for distinguishing mechanisms (in addition to the other evidence that may assist a differential diagnosis such as the plausibility of stress including adverse clinical signs and surrogate markers of elevated glucocorticoid action in the body such as atrophy of the thymus as previously mentioned). However, a useful indicator of adrenocortical toxicity versus stress is dependent on the magnitude of the adrenal hypertrophy, in turn dependent on the duration and magnitude of ACTH stimulation, and potent adrenocortical enzyme inhibitors resulting in sustained unopposed ACTH secretion, such as cyanoketone, can cause a 100% increase in adrenal weights in 3 days [47] a magnitude rarely seen with stress. Continuous ACTH infusion, mimicking the state of complete adrenocortical glucocorticoid inhibition and unopposed ACTH secretion, has been reported to result in a 10-fold increase in adrenal weight in 18 days [5,48]. Mechanistic models of assessing adrenocortical function, both in vivo models exploiting the action of ACTH in adrenocortical function, and in vitro systems for identifying specific sites and targets of toxicity, are discussed later. 4.3. Wildlife The manifestation of adrenocortical toxicity in wildlife is more difficult to recognise in the field but follows the same principles as laboratory animals. It is now becoming apparent that birds [49], fish [8] and marine mammals [9] are showing evidence of impaired adrenocortical function, ill health and reduced survival fitness linked to environmental chemical exposures. Recently, a population of dolphins exposed to the Deep Water Horizon oil spill pollution have been shown to have impaired cortisol and aldosterone secretion [9] compared with populations in nonpolluted areas. Aquatic species are often considered sentinel species for other wildlife but also the general health of the environment in which humans may also be exposed directly or indirectly. Chemical residues entering the food chain are well known to accumulate and form secondary exposure routes. Impairment of adrenocortical function at the population level may be expected to reduce survival fitness and may simply manifest in non-specific population declines. At the individual level the inability to adapt physiologically to adverse environmental insults may contribute to increased vulnerability, morbidity and mortality. The multitude of chemicals known to affect adrenocortical function via different target sites in the steroidogenic pathway may also act in concert: cumulative low level exposures may be as mixtures, or sequential exposures to different agents, increasing risk of adverse outcomes. There is a literature gap on the potential effects of low level mixtures and the dynamic physiological nature of the HPA axis raises the possibility that chemical exposures may result in occult subclinical blunting of the stress response, rather than frank abolition of glucocorticoid production, which would nevertheless impair survival fitness.
5. Models of adrenocortical assessment 5.1. In vivo approaches to adrenocortical functional evaluation As mentioned previously it is not possible to distinguish a mechanism from the observation of adrenocortical hypertrophy alone but it is important to prove functionality of the adrenal cortex when this finding is seen and to distinguish stress from adrenal toxicity (stress should result in an elevated glucocorticoid response to elevated endogenous ACTH and not result in impaired adrenocortical function). A simple test is to conduct an ACTH challenge study where injection of ACTH should provoke the secretion of glucocorticoid: the concept is in proving functionality of the adrenal cortex. As adrenocortical toxicants/enzyme inhibitors impair steroidogenesis, ACTH challenge in the species showing the effect will produce a blunted or lower response compared with controls. Potent adrenocortical enzyme inhibitors can produce marked inhibition of steroidogenesis after single or a few repeat exposures (e.g. etomidate [46] and cyanoketone [47]) and thus, the study can be relatively short duration. Designs can include positive controls but the basic protocol is to administer the test drug at a dose known to result in the adrenal effect and after a defined number of days administer ACTH, take blood samples at defined time points thereafter and measure the adrenocortical response, corticosterone levels in rats or mice and cortisol in dogs etc. This is a routine diagnostic test in human and veterinary clinical endocrinology to diagnose adrenocortical insufficiency conditions such as Addison’s disease and its variants. This test has been previously proposed to have utility in regulatory toxicology and adrenal endocrine disruption research [2,3,5,12,15]. By measuring additional steroid profiles it may also be possible to establish where in the pathway the drug/chemical may be exerting its effect. For example, if progesterone secretion is also reduced, it may be inferred that the inhibition is upstream and may therefore be 3-hydroxysteroid dehydrogenase D4,5 isomerase, CYP11A1 (cholesterol side chain cleavage), StAR or even down regulation of the ACTH receptor itself (shown to occur with aminoglutethimide [16]). However, the best tools for examining molecular sites of toxicity are cell lines. 5.2. In vitro adrenocortical model: H295R cell line Currently, the best available in vitro cell based model for studying adrenocortical function is the H295R cell line [3,15,50,51]. The H295R cell line is derived from a human adrenocortical carcinoma considered to arise from the border of the zona glomerulosa and zona fasciculata since it has the capability to produce both aldosterone and cortisol. Indeed it has virtually universal steroidogenic capability and thus it can be used to assess the entire process of steroidogenesis and it is now a validated test for androgen production in the USEPA battery of endocrine disruptor screening tests. This cell line does not express the ACTH receptor as well as normal cells specifically derived from the zona fasciculata, probably due to the phenotypic influence of its zona glomerulosa origin, and thus ACTH challenge is less effective and forskolin is used [51] which mimics the action of ACTH on signal transduction via cAMP. A typical protocol is to culture the cells in the presence of a number of test concentrations of the drug or chemical, challenge with forskolin and measure the resultant steroid secretion, aldosterone or cortisol. Evaluating steroid profiles (e.g. [40,44]) can provide information on the site of toxicity, but specific enzymes [30,43–45] and gene expression [19,32,45] can be directly measured. Fuller discussions of the utility of in vitro models and specifically the utility of the H295R cell line can be found in [15,50,51]. However, Harvey et al. [2,3,15] have suggested that this would be useful to develop and validate
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for the assessment of cortisol and aldosterone secretion which is currently lacking in any regulatory endocrine disruption strategy. Indeed, the H295R cell line continues to be enhanced. The use of gas chromatography tandem mass spectrometry analysis of H295R media has been developed, and used to evaluate steroid profiles, and compared with metabolomics methodology using ultra performance liquid chromatography–time-of-flight–mass-spectrometry (UPLC–ToF–MS) to identify specific “fingerprints” of endocrine disrupting chemicals (see [52,53]). It has also been proposed to use this cell line, along with other assays, in an integrated testing strategy for oestrogenicity to broaden sensitivity in identification of endocrine disruption through this mechanism [54]. Although the endpoints reported in these studies [52–54] pertain largely to sex steroids, they can also be applied to develop fingerprints using cortisol, aldosterone and other steroids specific to adrenocortical function. Such a development to the OECD Test Guideline on H295R in endocrine disruption evaluation [55], which currently only considers production of 17b-oestradiol and testosterone as endpoints, and validation of cortisol and aldosterone as endpoints, would be a major step in recognizing the unique role of the adrenal cortex and the extent to which glucocorticoid and/or mineralocorticoid producing adrenocortical cells may be a target for endocrine disruption. 6. Conclusion Evidence suggests that the adrenal cortex is a common toxicological target for both drugs and chemicals and inhibition of steroidogenic pathways is considered the major mechanism of action. The range of chemicals shown to inhibit adrenocortical steroidogenesis is diverse and there are multiple examples demonstrating that every step and enzyme in mineralocorticoid and glucocorticoid pathways can be affected. Human adrenocortical conditions, such as adrenogenital and salt wasting syndromes in infants, have defined molecular aetiologies related to problems with adrenocortical steroidogenic enzyme deficiencies [56–58] raising the possibility that environmental chemical exposure may provide a risk factor in promoting phenotypic variants of the disease at variable severities. The physiological role of the adrenal cortex can also hide expression of toxicity which may only become apparent when significant activation of the stress response is required to tolerate trauma and crises and assist survival. The lack of standardised adrenocortical functional testing is considered a major deficiency in regulatory endocrine disrupter strategies (see [2,3,59]) and more surprising given that the adrenal is the most common toxicological target in the endocrine system [4] its role as a vital organ and in developmental processes. Further, the tendency to “write-off” adrenal findings as stress in mammalian regulatory toxicology studies, in the absence of corroboratory evidence of adrenocortical functionality, is common and potentially dangerous which requires recognition in regulatory toxicology. Of most importance is that stress is not the only cause of adrenal hypertrophy in vivo: partial adrenocortical steroidogenic inhibition may co-exist with general toxicity induced stress, or operate independently, with expression being a function of relative drug/ chemical potency, dose and duration of exposure. It has been suggested that all cases of adrenocortical hypertrophy warrant further mechanistic investigation [5] and that a plausible supportive case based on additional evidence of adrenocortical competency should be made for all claims of a stress mechanism. The measurement of glucocorticoids is diagnostic, and the distinguishing feature between adrenocortical hypertrophy induced by stress versus steroidogenic toxicity is the adequacy of the glucocorticoid response. This can be assessed in vivo in the ACTH challenge study or in vitro in the H295R cell line. Finally, although
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controlled by the renin-angiotensin system rather than ACTH, the competency of production of aldosterone is also considered of major importance and it is suggested that this is also assessed in all cases of drugs or chemicals causing adrenocortical hypertrophy.
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