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Review
Adrenal toxicology: a strategy for assessment of functional toxicity to the adrenal cortex and steroidogenesis
Philip W. Harvey,* David J. Everett and Christopher J. Springall
Covance Laboratories UK Ltd, Toxicology Department, Otley Road, Harrogate, North Yorkshire, UK HG3 1PY Received 13 November 2006; Revised 12 December 2006; Accepted 13 December 2006
ABSTRACT: The adrenal is the most common toxicological target organ in the endocrine system in vivo and yet it is neglected in regulatory endocrine disruption screening and testing. There has been a recent marked increase in interest in adrenal toxicity, but there are no standardised approaches for assessment. Consequently, a strategy is proposed to evaluate adrenocortical toxicity. Human adrenal conditions are reviewed and adrenocortical suppression, known to have been iatrogenically induced leading to Addisonian crisis and death, is identified as the toxicological hazard of most concern. The consequences of inhibition of key steroidogenic enzymes and the possible toxicological modulation of other adrenal conditions are also highlighted. The proposed strategy involves an in vivo rodent adrenal competency test based on ACTH challenge to specifically examine adrenocortical suppression. The H295R human adrenocortical carcinoma cell line is also proposed to identify molecular targets, and is useful for measuring steroids, enzymes or gene expression. Hypothalamicuitary-adrenal endocrinology relevant to rodent and human toxicology is reviewed (with an emphasis on multi-endocrine axis effects on the adrenal and also how the adrenal affects a variety of other hormones) and the endocrinology of the H295R cell line is also described. Chemicals known to induce adrenocortical toxicity are reviewed and over 60 examples of compounds and their confirmed steroidogenic targets are presented, with much of this work published very recently using H295R cell systems. In proposing a strategy for adrenocortical toxicity assessment, the outlined techniques will provide hazard assessment data but it will be regulatory agencies that must consider the significance of such data in risk extrapolation models. The cases of etomindate and aminoglutethimide induced adrenal suppression are clearly documented examples of iatrogenic adrenal toxicity in humans. Environmentally, sentinel species, such as fish, have also shown evidence of adrenal endocrine disruption attributed to exposure to chemicals. The extent of human sub-clinical adrenal effects from environmental chemical exposures is unknown, and the extent to which environmental chemicals may act as a contributory factor to human adrenal conditions following chronic low-level exposures will remain unknown unless purposefully studied. Copyright @ 2007 John Wiley & Sons, Ltd.
KEY WORDS: adrenal; toxicity; adrenocortical; protocol; steroidogenesis; H295R; rat; human; cortisol; corticosterone; StAR; endocrine disruption; medulla
Introduction
The adrenal is arguably the neglected organ in endocrine toxicology and the lack of recognition of the importance of adrenal function in a regulatory endocrine disruption context, and the need for an adrenal toxicology assess- ment strategy, has been pointed out (Harvey and Johnson, 2002; Harvey and Everett, 2003, 2006; Hinson and Raven, 2006; Oskarsson et al., 2006). This is surprising given the functional role of the adrenal in health, metabolism, development and in the range of hormones it produces, and that it has been documented to be the
most common toxicological target of all endocrine organs (Ribelin, 1984; Colby and Longhurst, 1992; Rosol et al., 2001). Indeed, in surveys based on chemically induced endocrine lesions seen in in vivo toxicology studies, the order of endocrine organ toxicity by frequency of reported effects was adrenal > testes > thyroid > ovary > pancreas > pituitary > parathyroid (e.g. Ribelin, 1984; Colby and Longhurst, 1992) with the adrenal cortex, rather than the medulla, being the most frequent site of toxicity within the adrenal gland. Harvey (1996a, b) and Harvey et al. (1999) discuss primary and secondary/ indirect toxicological responses relevant to the adrenal and also the importance of the adrenal in the overall response to, and tolerance of, toxic insult.
The adrenal is a vital organ where chemical inhibition of normal function of the cortex has been documented to have lethal consequences in humans from single brief
* Correspondence to: Philip W. Harvey, Covance Laboratories UK Ltd, Toxicology Department, Otley Road, Harrogate, North Yorkshire, UK HG3 1PY.
exposures to some compounds, at remarkably low dose levels. Indeed glucocorticoid production is the single most important physiological response for survival of an organism post infection or injury (e.g. Munck et al., 1984). For example, the cases of etomidate (an anaes- thetic induction agent administered intravenously as a single dose of approximately 0.3 mg kg-1 bodyweight) and aminoglutethimide, formerly used as a weak anti- convulsant and sedative but now used in metastatic breast cancer and Cushings syndrome because of effects on steroidogenesis, caused cases of adrenocortical sup- pression unpredicted and unrelated to the primary pre- scribed action of the compound, and in the case of etomidate, rapid patient death through Addisonian crisis (Leddingham and Watt, 1983; Leddingham et al., 1983; Goldberg, 1983; Raven and Hinson, 1996; Hinson and Raven, 2006; Vermeulen et al., 1983). Etomidate proved to be a potent and selective 11/18 hydroxlase (cyto- chrome P450 11B1; CYP11B1; CYP110/18) inhibitor blocking cortisol production, and although its medicinal use is clearly a worst-case human exposure scenario, the fact that it has prolonged adrenocortical suppressant activity at low doses (in the ug kg-1 body weight dose range) after only a single dose/exposure, raises the ques- tion of whether low-level long-term exposures of the human population to environmental chemicals could also produce unrecognised adrenal effects of various degrees. As cortisol production can be affected by chemical action at a number of sites along the steroidogenic pathway (Harvey and Johnson, 2002; Harvey and Everett, 2003) combined exposures to chemicals or mixtures, potenti- ally affecting different sites along a common pathway, may precipitate adrenocortical dysfunction at even lower exposure rates. The lack of a regulatory framework to study adrenal toxicity means that there is a relative paucity of quality standardised data, that data acquisition is slow, and there are no recommended standardised regulatory procedures/protocols for assessment to remedy this situation.
As well as directly operating on the adrenal cortex, certain drugs can suppress adrenocortical function by inhibition of hormones higher in the endocrine axis at the level of the hypothalamus or pituitary, but the end result of deficits in glucocorticoid secretion are the same as a direct-acting adrenocortical enzyme inhibitor. For example, valproic acid, bromocriptine, cyproheptadine, ketanserin, ritanserin, somatostatin analogues and glu- cocorticoids can suppress pituitary adrenocorticotrophic hormone (ACTH) secretion, and in turn adrenal glu- cocorticosteroid secretion as the endpoint, in both humans and rats; valproic acid also suppresses hypothalamic corticotrophin releasing hormone (e.g. Mercado-Asis et al., 1997; Tringali et al., 2004; Kasperlik-Zaluska et al., 2005; Sonino et al., 2005). The importance of this is that these compounds suppress the adrenal by a mechanism only likely to be detected by in vivo studies,
with intact hypothalamicuitary-adrenocortical (HPA) axis function, which in turn affects any proposed assess- ment strategy for adrenal function (see later).
Previous reviews have collectively introduced know- ledge of the range of structurally diverse compounds known to induce toxicity in vivo to the adrenal cortex (e.g. Ribelin, 1984; Szabo and Lippe, 1989; Colby and Longhurst, 1992; Colby, 1996; Raven and Hinson, 1996; Hinson and Raven, 1999; Rosol et al., 2001) and to the adrenal medulla (Tucker, 1996; Hinson and Raven, 1999; Rosol et al., 2001) but the potential molecular basis of such effects has only more recently emerged and this, and toxicologically relevant endocrinology, is discussed later. Identified factors predisposing the adrenal to toxic insult in vivo include: the large number of potential toxicological targets such as receptors, enzymes and peripheral hormone carrier molecules; high vascularity and disproportionately large blood volume received per unit mass; the high content of unsaturated fatty acids in adrenocortical cell membranes susceptible to lipid peroxidation; lipophilicity due to rich cholesterol and steroid content; and the high content of cytochrome P450 (CYP) enzymes present in the adrenal cortex, that norm- ally catalyse steroidogenesis, but which can also produce reactive metabolites of toxicants and hydroxylation reac- tions that may generate free radicals (e.g. Hinson and Raven, 2006). Although these mechanisms are largely relevant to in vivo adrenal toxicology and consequent pathology, effective and relevant in vitro models, such as the H295R cell line, have only very recently been devel- oped and these are now expanding the range of known adrenocortical toxicants and molecular mechanisms of action (e.g. Ohno et al., 2002; Sanderson et al., 2002, 2004; Li et al., 2004; Hilscherova et al., 2004; Voets et al., 2004; Zhang et al., 2005; Kau et al., 2005; Muller- Vieira et al., 2005; Li and Wang, 2005; Blaha et al., 2006; Lin et al., 2006; Xu et al., 2006; Hecker et al., 2006; Gracia et al., 2006; Canton et al., 2006; Imagawa et al., 2006; Oskarsson et al., 2006; Sanderson, 2006; see discussion in Harvey and Everett, 2003).
The purpose of this paper is to review developments in adrenocortical toxicology and provide a standardised experimental strategy for the investigation of toxicolog- ical effects on adrenocortical function and mechanism elucidation, and importantly, one that could be applied in a regulatory toxicology context. Specific adrenal end- ocrinology of laboratory rodents is reviewed, particularly the diverse endocrinological influences on the adrenal by hormones such as prolactin and oestradiol, and dif- ferences with humans highlighted. Human adrenal dys- function is reviewed giving examples of conditions that could also be chemically induced, and the molecular basis of action of known adrenocortical toxicants is discussed together with a strategy and suggested pro- tocols for examining adrenal toxicity. The danger of explaining adrenal findings in regulatory toxicology
studies as ‘stress-related’, particularly adrenal hyper- trophy in rodents, in the absence of clear evidence of adrenocortical competence and glucocorticoid secretion, is also discussed.
Human Adrenal Dysfunction
There are numerous well-documented conditions affect- ing humans and because these involve alterations to the control and function of various adrenocortical steroido- genic and enzyme pathways, it is conceivable that such effects may also be chemically induced, for example through enzyme inhibition. Chemical inhibition of key steroidogenic enzymes is reviewed later. However, by far the most toxicologically significant and frequent effect is adrenocortical suppression iatrogenically induced by drugs (e.g. Leddingham and Watt, 1983; Goldberg, 1983; Raven and Hinson, 1996; Hinson and Raven, 2006; Vermeulen et al., 1983) and particularly the corti- costeroids used for anti-inflammatory action (e.g. Kaliner, 2006). Chemicals that inhibit the enzymes along the cortisol biosynthetic pathway may induce adrenocortical insufficiency and many chemicals have been shown to inhibit adrenocortical steroidogenic enzyme targets (discussed later).
Adrenogenital syndrome is a congenital condition where a fault in enzyme activity in the adrenal pro- duces an excess of androgen secretion which virilises/ masculinises females during development. The ability to manipulate the enzymes leading to this condition (Hakki and Bernhardt, 2006) raises the possibility that chemical toxicity may cause this syndrome. Similarly, salt-losing congenital adrenal hyperplasia, resulting in adrenal crisis and cardiovascular collapse in infants, typically presents within the first two weeks of life. However a recent report has shown that this can also occur relatively late, outside the usually expected time frame, in 6-8 month old children as a result of defects in steroidogenic acute regulatory protein (StAR) function, which is apparently much slower to express (Gassner et al., 2004). Gassner et al. (2004) reported that two out of the three patients studied had mutations in the StAR gene, and no muta- tions were found in a third suggesting a ‘novel disease’ and as no mutation was found, transient environmental or chemical factors may be implicated. Chemical inhibition of StAR function is discussed later. These examples illus- trate the developmental vulnerability of the adrenal and consequences of altered secretory function during critical early life stages, and the above example of salt-losing congenital adrenal hyperplasia may involve at least three different sub-types.
Cushings syndrome is a condition of over-production of adrenocortical steroids, particularly glucocorticoids, attributed in humans to over-expression of the adrenal adrenocorticotrophic hormone (ACTH) receptor (Clark
and Metherell, 2006) and is also a common side effect of glucocorticoid therapy (Newell-Price et al., 2006). This condition is unlikely to be encountered in regulatory toxicology of compounds not structurally related to glu- cocorticosteroids, although drugs such as caffeine and other agents that amplify cAMP can cause marked hypothalamicuitary-adrenal (HPA) axis stimulation in rodents (Garside and Harvey, 1992; Hadley et al., 1990; Spindel et al., 1983) and elevated endogenous gluco- corticoids may have a modest effect. Interestingly, whilst excess glucocorticoids in humans typically produces increased weight gain (e.g. Frank et al., 2004), studies of repeat dose administration of corticosterone in the rat typically show reductions in body weight gain (Harvey et al., 1992) illustrating species differences in response, although reduction of muscle mass and protein catabo- lism is consistent.
Priorities of Assessment in Adrenal Toxicology
In the context of adrenal/endocrine toxicology, the major priority should be to identify compounds causing func- tional suppression of the adrenal, since there are already clear examples of chemically induced adrenal suppression in human clinical toxicology. In regulatory toxicology tests, adrenocortical inhibition/suppression may manifest as an enlarged adrenal gland particularly in rodents. However, adrenal weights can be relatively insensitive depending on study design and the organ to brain weight ratio appears to be most predictive for adrenal weights (Bailey et al., 2004). In this case, the rodent adrenal may be enlarged because of continuous ACTH stimulation because the adrenal cortex is not producing corticosterone to provide negative feedback regulation of pituitary ACTH release (Harvey and Everett, 2003, 2006).
Adrenal enlargement is an indicator of ACTH stimula- tion and may occur as a result of classical ‘stress’ or pharmacological stimulation of the HPA axis, as well as loss of feedback inhibition, but there are usually other indicators to identify that the enlargement is not due to adrenocortical inhibition/suppression. In standard regulatory toxicology protocols that do not assess end- ocrinology, these involve other evidence of adrenal function and corticosteroid secretion and action, such as reduction in body weight gain, atrophy (involution) of the thymus, glycogen status of the liver, and if clinical chemistry evaluations are conducted at appro- priate times relative to dosing, the classical effects of glucocorticoids may be detected in blood glucose and other measurements. There are few toxicology studies of the effects of corticosterone in the rat, but Harvey et al. (1992) reported that in a 1-month toxicology study of corticosterone administration producing high but phy- siologically relevant blood corticosterone concentrations
approximating stress values, reduced body weight gain coupled with lower thymus, prostate and seminal vesicle weights were findings attributed to both the direct effects of corticosterone (body weight and thymus weight) and the inhibition by corticosterone of luteinizing hormone and testosterone (Kamel and Kubajak, 1987; Srivastava et al., 1993; Sankar et al., 2000). This combination of findings (reduced body weights, lower thymus, prostate and seminal vesicles weights indicative of the effects of corticosterone) in combination with enlarged adrenal (indicative of ACTH stimulation), may prove useful markers of adrenocortical steroid production, and there- fore adrenocortical competency, in the absence of direct data such as blood corticosterone concentrations. Micro- scopic histopathology evaluation of thymus, spleen and other glucocorticoid sensitive tissues can also assist to establish the recent functional history of the adrenal. Conversely, small adrenals/adrenal atrophy is indicative of a loss of trophic support of the adrenal by ACTH, and this too may result in deficits in functional capability of the cortex to produce glucocorticoids.
An enlarged adrenal in the absence of evidence of adrenal functional competency should not in isolation be considered to be a ‘simple’ stress related effect, and therefore of limited toxicological significance, and possi- ble functional suppression should be investigated further. Functional suppression of glucocorticoid output, via inhi- bition of any of the steps in the steroidogenic pathway, or inhibition of pituitary ACTH or indeed hypothalamic function, is considered a potentially serious toxicological finding of direct relevance to humans. Experimental strat- egies to investigate adrenocortical suppression are dis- cussed later. The majority of molecular biosynthetic steroidogenic steps within the adrenal cortex are generally similar between the rat, mouse and human supporting the usefulness of the rodent as a predictive toxicolog- ical model. However, factors triggering activation and sensitivity of the HPA axis vary, and the dominant glucocorticosteroid in rodents is corticosterone, compared with cortisol in humans and other higher mammals.
Although pituitary-adrenal activation through direct or indirect/stress related mechanisms is considered a much less serious finding, if encountered in regulatory toxicology studies, compared with frank adrenal suppres- sion, pituitary-adrenocortical overactivity nevertheless can be detrimental in terms of developmental toxicity. ACTH stimulated adrenal steroids can cross the placenta and affect fetal HPA development in rodents, including atrophy of the fetal adrenal through functional sup- pression of the developing HPA system (Skebelskaya, 1968; Milkovic et al., 1976). Recent studies on perinatal glucocorticoid exposure have shown a variety of per- manent effects in adulthood, for example molecular (annexin-1), functional and morphological changes in the anterior pituitary (Theogaraj et al., 2005) and in host defence cells in blood and lung (Theogaraj et al., 2006),
which extends glucocorticoid modulation of immune function to developmental immunotoxicology (see also Spinedi et al., 2005 for neuroendocrine-immune inter- actions specifically involving the HPA axis). These recent examples further illustrate that inappropriate glucocorticoid exposure in critical periods can have far-reaching and apparently irreversible adverse devel- opmental effects, and alter physiological programming of glucocorticoid-sensitive tissues in endocrine and immune systems. Baldwin (1996) reviews the role of natural and synthetic glucocorticosteroids in development and devel- opmental toxicity in laboratory species and humans.
Adrenal endocrinology relevant to toxicology
Reviews of the general endocrinology of the HPA axis and ACTH secretion (Buckingham et al., 1992) and hormone synthesis, metabolism, transport and action (related to toxicology) are provided elsewhere (Hinson and Raven, 1996; Raven and Hinson, 1996; Gumbleton and Nicholls, 1996; Hinson and Raven, 1999). The major activator of the HPA axis is stress, which may be psy- chological or physical such as cold, exercise, anaesthesia, infection, hypoglycaemia, injury or indeed toxic insult. The release of ACTH from the pituitary is controlled by hypothalamic corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP). ACTH is released into the blood to stimulate the adrenal cortex to produce and secrete glucocorticoids (corticosterone in rodents and cortisol in higher mammals and humans). The glu- cocorticoids produce negative feedback inhibition at the level of both pituitary and hypothalamus to reduce ACTH output.
There are other hormonal factors relevant to the endocrinology of the HPA axis. Oestrogen is known to stimulate ACTH (Barrett, 1960) and therefore adreno- cortical function, whilst progesterone is reported to inhibit adrenocortical function in the rat (Rodier and Kitay, 1974). Gonadotrophins, melanocyte stimulating hormone and prolactin both alone and synergistically with oestro- gen, have been reported to stimulate corticosterone in the rat (Sugihara et al., 1982; Vinson et al., 1976; Vasquez and Kitay, 1978; Ogle and Kitay, 1979). Prolactin and growth hormone are reported to stimulate chromaffin cells in the adrenal medulla and produce hyperplasia (Rosol et al., 2001; Colby and Longhurst, 1992). Further, endogenous glucocorticoids have a number of endocrino- logical effects other than controlling ACTH release, and are reported to suppress prolactin in rats (Gala et al., 1981) and humans (Bratusch-Marrain et al., 1982), acutely inhibit insulin in rats (Billaudel and Sutter, 1982) and suppress thyroid stimulating hormone (Pamenter and Hedge, 1980). Corticosterone also inhibits luteinizing hormone and testosterone in vivo and in vitro in the male
rat (Kamel and Kubajak, 1987; Srivastava et al., 1993; Sankar et al., 2000) and the reduction in testosterone can result in decreased prostate and seminal vesicle weights (Harvey et al., 1992). The purpose of reviewing these classical endocrinology effects is to draw attention to the fact that influences on adrenal function can occur in a number of diverse ways, and altered adrenal function may have far reaching repercussions physiologically, not least to the function of thyroid, gonads and pancreas. Understanding the endocrinology of rodents is important for understanding mechanisms of toxicity in regulatory studies, and adrenal toxicity may be direct or secondary to altered endocrine function and occur within and across the HPA axis (e.g. Harvey, 1996a, 1996b; Harvey et al., 1999).
Chemicals known to cause adrenal toxicity
There are a number of previous reports that provide ex- amples of chemicals with known adverse effects on the adrenal gland, both cortex (e.g. Ribelin, 1984; Szabo and Lippe, 1989; Colby and Longhurst, 1992; Colby, 1996; Rosol et al., 2001) and medulla (e.g. Tucker, 1996; Rosol et al., 2001) largely derived from results of in vivo studies and descriptive histopathological lesions, with the cortex being the most frequently affected site. It is
only relatively recently that the molecular basis of the adrenocortical findings has been researched, with the focus being on the complex process of steroidogenesis and elucidation of molecular mechanisms of action.
Figure 1 shows the steroidogenic pathways in the adrenal cortex, identifying potential molecular targets for toxic action. Table 1 lists over 60 chemicals affect- ing adrenal function and steroidogenesis and their reported molecular targets including StAR, and CYP and dehydrogenase enzymes. The number of individual com- pounds reported in the literature is larger when com- pound class analogues and metabolites from the cited examples are also included, or when data on common steroidogenic targets in non-adrenal cells are included, and in this case the in vitro molecular regulation of the target in adrenal and non-adrenal cells (e.g. StAR) may not be identical between cell lines but vulnerability can be deduced. Much of this recent work has used in vitro tests, such as the human adrenocortical carcinoma derived NCI-H295R cell line systems. Techniques range from biochemical hormone and enzyme assays to gene expres- sion studies and the utility of the H295R cell line in adrenocortical toxicology is specifically reviewed in the next section.
There are fewer examples of chemicals affecting the medulla and Tucker (1996) reviews the chemicals re- ported in the literature to induce non-proliferative lesions
CHOLESTEROL
StAR Protein
Mitochondrial membrane
CHOLESTEROL
Androstenediol-17x
Androstenediol-17฿
1
10
1
9
Pregnenolone
3
17-hydroxy pregnenolone
4
Dehydroepiandrosterone
2
2
2
PROGESTERONE
3
17-hydroxy progesterone
4
9
Androstenedione
TESTOSTERONE
5
5
8
8
11-deoxycorticosterone
11-deoxycortisol
Oestrone
9
OESTRADIOL
7
6
11-dehydro corticosterone
6
11
ALDOSTERONE
11
CORTISOL
Cortisone
CORTICOSTERONE
StAR = Steroid Acute Regulatory protein. Key to enzymes: 1 = CYP11A1 (cholesterol side chain cleavage).
2 = 3ß-hydroxysteroid dehydrogenase-4 4,5 isomerase. 3 = CYP17 (17a-hydroxylase).
4 = CYP17 (17, 20 lyase). 5 = CYP21 (21 hydroxylase). 6 = CYP11B1 (11}/18-hydroxylase).
7 = CYP11B2 (aldosterone synthase). 8 = CYP19 (aromatase). 9 = 17ß-hydroxysteroid dehydrogenase.
10 = 17a-hydroxysteroid dehydrogenase. 11 = 11ß-hydroxysteroid dehydrogenase.
Figure 1. Adrenocortical steroidogenesis pathways
| Steroidogenic target | Compound | Reference |
|---|---|---|
| Steroid Acute regulatory (StAR) protein CYP11A1 (CYPscc) | Econazole, miconazole, lindane | Walsh et al. (2000a); |
| Glyphosate Dimethoate Carbachol Ethanol Arsenite, anisomycin Bromocriptine Spironolactone | Walsh and Stocco (2000); Oskarsson et al. (2006) Walsh et al. (2000b) Walsh et al. (2000c) Janossy et al. (2001) Khisti et al. (2003) Zhao et al. (2005) Kan et al. (2003) Hilscherova et al. (2004) | |
| Aminoglutethimide Dimethoate Bromocriptine | Camacho et al. (1967); Vermeulen et al. (1983); Johansson et al. (2002); Hecker et al. (2006) Walsh et al. (2000c) Kan et al. (2003) | |
| CYP17 | Spironolactone Ketoconazole | Kossor et al. (1991) Loose et al. (1983); Johansson et al. (2002) |
| Flavonoids (6-hydroxyflavone) PCB126 Penta, octa, deca-brominated diphenyl ethers, tetrabromobisphenol-A, hexabromocyclododecane isomers Thiazolidinediones-pioglitazone | Ohno et al. (2002) Li and Wang (2005) Canton et al. (2006) Kempna et al. (2006) | |
| 3-Hydroxysteroid dehydrogenase A 4,5 isomerase | Cyanoketone Trilostane 6-Hydroxyflavone, daidzein, genistein, biochanin A, formononetin PCBs (101, 110, 126, 149) PAHs/PCBs Thiazolidinediones-pioglitazone | McCarthy et al. (1966) Potts et al. (1978) Ohno et al. (2002) Xu et al. (2006) Blaha et al. (2006) Kempna et al. (2006) |
| 17ß-hydroxysteroid dehydrogenase CYP21 CYP11B1 (CYP11B/18) | Di (2-ethylhexyl) phthalate PCBs (101, 110, 126, 149) | Akingbemi et al. (2001) Xu et al. (2006) |
| RU486 Ketoconazole Flavonoids PCB126 PAHs/PCBs | Albertson et al. (1994) Johansson et al. (2002) Ohno et al. (2002) Li and Wang (2005) Blaha et al. (2006) | |
| Metyrapone Mitotane (o,p-DDD); MeSO2-DDE Etomidate | Liddle et al. (1958); Johansson et al. (2002) Hornsby (1989); Lindhe et al. (2002); Johansson et al. (2002) Leddingham and Watt (1983); Hinson and Raven (1996) | |
| Ketoconazole; aminoglutethimide Flavonoids PCB126 | Johansson et al. (2002) Ohno et al. (2002) Li and Wang (2005); Lin et al. (2006) | |
| PCBs (101, 110, 126, 149) Efonidipine, mibefradil | Xu et al. (2006) Imagawa et al. (2006) | |
| CYP19 (Aromatase) | Prochloraz, imazalil | Andersen et al. (2000) |
| Diindolylmethanes Triazines-atrazine, simazine, propazine Di-, tributyl and phenyltin chlorides Imidazoles, vinclozalin, fenarimol Flavonoids (7-hydroxyflavone, chrysin) Fadrozole PCBs (101, 110, 126, 149) | Sanderson et al. (2001b) Sanderson et al. (2001a) Sanderson et al. (2002) Sanderson et al. (2002) Sanderson et al. (2004) Muller-Vieira et al. (2005); Hecker et al. (2006) Xu et al. (2006) |
| Steroidogenic target | Compound | Reference |
|---|---|---|
| CYP11B2 (Aldosterone synthase) | Guanabenz-related amidinohydrazones | Soll et al. (1994); |
| Hinson and Raven (1996) | ||
| PCB126 | Li and Wang (2005); Lin et al. (2006) | |
| Fadrozole | Muller-Vieira et al. (2005) | |
| PCBs (101, 110, 126, 149) | Xu et al. (2006) | |
| Efonidipine, mibefradil PAHs/PCBs | Imagawa et al. (2006) Blaha et al. (2006) | |
| Altered steroid output, enzyme activity and gene expression in H295R cells | Examples of steroid studies | |
| Ketoconazole, prochloraz, fadrozole, aminoglutethimide, vinclozalin | Hecker et al. (2006) | |
| (Loestradiol, Îprogesterone, Î pregnenolone, ¿testosterone: profile varies with chemical) 6-Hydroxyflavone, 4-hydroxyflavone, apigenin, daidzein, genistein, formononetin (cortisol, \DHEA) | Ohno et al. (2002) | |
| Procaine (\HMG-coA/cholesterol) | Xu et al. (2003) | |
| Efonidipine (\cortisol, Valdosterone) | Imagawa et al. (2006) | |
| Fadrozole (\cortisol, Vandrogens, Valdosterone) | Muller-Vieira et al. (2005) | |
| Digoxin, ouabain (\cortisol, Valdosterone) | Kau et al. (2005) | |
| Lindane (\cortisol) | Oskarsson et al. (2006) Li et al. (2004) | |
| PCB126 (Îaldosterone) | ||
| PCB126 (Îaldosterone, Îcortisol, Vandrogens) | Li and Wang (2005) | |
| Examples of enzyme studies (see main table) PAHs/PCBs | Blaha et al. (2006) | |
| Triazines-atrazine, simazine, propazine | Sanderson et al. (2001a) | |
| Di-, tributyl and phenyltin chlorides; imidazoles, vinclozalin, fenarimol | Sanderson et al. (2002) | |
| Flavonoids | Sanderson et al. (2004); Ohno et al. (2002) | |
| Thiazolidinediones-pioglitazone, | Kempna et al. (2006) | |
| Examples of gene expression studies (expression of steroidogenic enzymes see main table) | Hilscherova et al. (2004); Zhang et al. (2005); Li and Wang (2005); | |
| Kau et al. (2005); | ||
| Gracia et al. (2006); Oskarsson et al. (2006); Kempna et al. (2006); Xu et al. (2006) |
Î increased hormone secretion, \ decreased hormone secretion.
and their proposed mechanisms (e.g. cysteamine hydro- chloride, acrylonitrile, tamoxifen, reserpine and mannitol) and also proliferative lesions (e.g. polyols, growth hormone). Phaeochromocytoma is a common finding in the rat, particularly the male, and studies of chromaffin cell proliferation suggest excess growth hormone or prolactin, stimulation of cholinergic nerves and dietary mechanisms such as hypercalcaemia (Tucker, 1996; Rosol et al., 2001) as causes. Conversely, whilst more agents historically have been reported to affect the adrenal cortex (Ribelin, 1984; Colby, 1996), implying direct adrenocortical toxicity and sensitivity/vulnerability compared with the medulla, the proportion showing true direct toxicity compared with secondary effects or stress due to, for example, administration of a compound at the maximum tolerated dose (MTD) is unknown. A wide variety of compounds have been shown to result in higher corticosterone secretion in rodents at dose levels approximating the MTD indicating stress-induction of the
HPA axis: this is reviewed in Harvey (1996a) and indeed administration of compounds at the MTD is by definition stressful (e.g. Miller, 1992). In these cases the reported elevations of corticosterone at least establishes function- ally competent adrenals.
Assessing Adrenal Toxicity: Establishing Adrenocortical Competency and Identifying Molecular Targets
The need to develop a toxicology strategy for the assessment of adrenal function and the process of steroidogenesis as targets for toxicity has been pointed out (Harvey and Everett, 2003, 2006; Oskarsson et al., 2006; Hinson and Raven, 2006; Sanderson, 2006) and a two phase strategy is suggested in the following dis- cussion. The strategy is based on a short in vivo study where the competence of the adrenal in treated rodents
is assessed by an ACTH challenge and the resulting corticosterone secretion is measured in the blood as an endpoint marker of adrenocortical function. As adreno- cortical impairment/suppression can be induced by inhibition of corticotrophin releasing hormone or ACTH (e.g. Mercado-Asis et al., 1997; Tringali et al., 2004; Kasperlik-Zaluska et al., 2005; Sonini et al., 2005), as well as by inhibiting adrenocortical steroidogenic en- zymes, an in vivo study in intact animals is required to test the integrity of the entire HPA axis by steroid secre- tion. The ability of untreated rats to respond to an ACTH challenge by markedly increased corticosterone secretion, compared with compound treated rats that can not, would provide strong evidence of adrenocortical suppression. The use of an in vitro system, such as the H295R human adrenocortical carcinoma cell line can then be employed as a second tier to elucidate mechanisms of action and provide evidence for, or exclude, direct adrenocortical effects such as altered StAR function, as distinct from enzyme inhibition.
Establishing Adrenal Competence: A Proposed In Vivo Study Design to Assess Adrenocortical Suppression by ACTH Challenge in Rodents
The in vivo adrenal functional competency test is pro- posed if there is evidence in the existing toxicology data of compound related adrenal change, such as adrenal histopathology or organ weight effects and is designed to confirm, or more importantly exclude, adrenocortical sup- pression. The proposed design consists of a control group to assess basal corticosterone and the stress-inducing effects of any procedures, a positive control group admin- istered ACTH challenge, and a number of test compound groups to provide information on dose response or no effect level as appropriate that are also challenged with ACTH. Doses of ACTH known to provoke corticosterone secretion in the rat are at least 15 IU kg-1 (e.g. Gamallo et al., 1992) or 50-500 µg kg-1 (e.g. Turner et al., 1998) administered subcutaneously. The test compound groups should probably be treated for 3-5 days before challenge to allow time for any potential inhibition of steroidogenic enzymes to occur. Blood samples are taken after ACTH challenge. The endpoint is whether compound treated rodents can respond to ACTH challenge to a similar degree as the positive controls in terms of blood corticosterone levels and other marker steroids or whether there is evidence of adrenocortical suppression or impair- ment. Test compound groups not treated with ACTH can also be evaluated to examine basal corticosterone levels and the design is flexible; if these rats were found initially to show reduced corticosterone, but then re- sponded to ACTH challenge, it may be concluded that they had a test compound induced deficit in hypothalamicuitary function and endogenous ACTH
production/secretion. Additionally, depending on study design, blood taken from the various groups can also be used to assess a variety of other hormones. It is prefer- able to measure all the hormones in a given endocrine axis, however, in the case of ACTH, blood should be sampled within 20 s of rat/cage disturbance or the experi- mental procedure itself affects ACTH levels. This test is also applicable to other laboratory species.
At the end of the study, adrenals and other tissues may be taken for further investigations such as immunohisto- chemistry or molecular studies. A recent study of the adrenal toxicity resulting from acute administration of 7,12-dimethylbenz[a]anthracene (DMBA) in rats, which caused apoptosis in the zona reticularis and zona fasciculata followed by severe haemorrhagic necrosis, reported the use of histopathological and immunohisto- chemical techniques, illustrating that additional tissue endpoints can be readily studied in retained adrenals (Fu et al., 2005). However, the benefit of measuring corticosterone is that it can be used as a rapid diagnostic biomarker without the need for histopathology. Fu et al. (2005) also measured serum corticosterone, which was predictably reduced in DMBA rats with severely necrotic adrenals. Interestingly, in evaluating enzyme activity in adrenals taken from rats after toxicological treatments, the functional reserve of the adrenal may be a factor affecting enzyme activity and the expression of enzyme target toxicity: ciprofibrate has been reported to reduce 3ß-hydroxysteroid dehydrogenase-isomerase, and to a lesser extent 17ß-hydroxysteroid dehydrogenase in the testes, but much less so in the adrenal (Hierlihy et al., 2006). This difference in tissue sensitivity was attri- buted to different enzyme control mechanisms, but 3ß- hydroxysteroid dehydrogenase-isomerase activity was 10-fold higher in the adrenal than the testes, indicating a greater reserve and resistance to inhibition. Under normal physiological conditions the adrenal cortex has a large functional reserve capacity, but inhibition of a key enzyme such as 110/18-hydroxlase can completely and rapidly abrogate this (see earlier discussion of etomidate, Fig. 1 and Table 1).
Special Considerations
· Rodents may require sham dosing and handling for 7- 10 days prior to study start to habituate and desensitise to the stress of handling and dosing procedures (gavage dosing of certain vehicles is reported to be stressful and increase corticosterone in rats - Brown et al., 2000). The pre-study phase may provide an opportunity to obtain basal corticosterone values for all animals.
· Stage of the oestrous cycle should be assessed if females are used, as this affects adrenocortical status (Ogle and Kitay, 1979; Nicholls and Chevins, 1981) as does oestrogen (Barrett, 1960) and progesterone (Rodier and Kitay, 1974), and group sizes need to be
adequate because of individual variability in hormones. If there are data confirming the absence of sex dif- ferences in pharmacokinetics or toxicology, the use of males only may be sufficient.
· Blood samples should be collected at the same time of day, within a narrow time window avoiding group bias, under minimal/controlled disturbance conditions. Care should be taken to house rodents in cages of identical numbers of animals and a cage size of three individuals is optimal to carry out timed procedures per cage. Blood sampling should ideally be carried out in a separated annex room.
· Extreme care needs to be taken to ensure genuine non-stressed control values. Blood sampling should be completed within 3 min of cage disturbance for cortico- sterone measurements in rodents (e.g. Nicholls and Chevins, 1981) and room noise etc controlled. Rodents should only be blood-sampled once or twice with sufficient time for recovery.
· Range finding work may be required in the strain of rat or mouse to select an effective ACTH challenge dose and time course after ACTH treatment for blood sam- pling for corticosterone estimation.
· A typical design would be to dose young adult rats (e.g. n = 6-9 per group) with test compound by oral gavage (or the route of human exposure) for 3-5 days, and on the challenge day dose test compound, and after 1 h administer the ACTH challenge by sub- cutaneous injection. Blood samples can be obtained at an appropriate interval after this (e.g. 1-3 h later if depot injection is used) to estimate plasma cortico- sterone, comparing data with both untreated and ACTH treated controls.
· Timing of dosing versus challenge and blood sampl- ing would depend on Cmax/Tmax of the test compound, for example, by the oral route. It is assumed that this information is known prior to conducting an in vivo adrenal suppression test, as systemic exposure is fundamental to this mechanism.
Identifying Molecular Targets and Mechanisms: In Vitro Assessment using H295R Human Adrenocortical Cells
In a regulatory toxicology and strategic evaluation context, the H295R cell line has been suggested as a potentially useful tool for examining toxicity both to the adrenal and also the process of steroidogenesis (Harvey and Everett, 2003, 2006; Oskarsson et al., 2006; Hinson and Raven, 2006; Sanderson, 2006). This cell line expresses all the key enzymes necessary for steroido- genesis and the production of all major steroids such as progesterone, androgens, oestrogens, glucocorticoids and the mineralocorticoid aldosterone (e.g. Zhang et al., 2005). Numerous recent studies have now evaluated this
cell line and methodology as a rapid in vitro screening and mechanism elucidation tool, specifically for toxicant induced effects on steroidogenesis (e.g. Hilscherova et al., 2004; Zhang et al., 2005; Muller-Vieira et al., 2005; Hecker et al., 2006; Gracia et al., 2006; Oskarsson et al., 2006) with the consensus that it is a relevant, suitable and sensitive system for evaluating mechanisms of adrenocortical function. H295R cell systems can be used to assess the effects of compounds on steroid production and secretion (e.g. Muller-Vieira et al., 2005; Hecker et al., 2006; Imagawa et al., 2006; Oskarsson et al., 2006; Voets et al., 2004) on steroidogenic enzyme activity (e.g. Ohno et al., 2002; Canton et al., 2006; Oskarrson et al., 2006) and for gene expression profiling of key steroidogenic genes (e.g. Hilscherova et al., 2004; Zhang et al., 2005; Gracia et al., 2006; Blaha et al., 2006; Xu et al., 2006; Oskarsson et al., 2006).
H295R cells have been well-described endocrinologic- ally, and have functional ACTH receptors (concentra- tions of which may be increased by certain chemical toxicants to sensitise the cells, e.g. Li and Wang, 2005), functional corticotrophin-releasing hormone (CRH) receptors (Willenberg et al., 2005) and respond to forskolin and isobutyl methylxanthine cyclic AMP induc- tion (Sanderson et al., 2002) and dibutyryl cyclic AMP by stimulated corticosteroid synthesis (Xu et al., 2003). They also respond to angiotensin II by production of aldosterone (Kau et al., 2005), vasoactive intestinal peptide (VIP) by production of cortisol (Nicol et al., 2004), have functional atrial natriuretic peptide (ANP) receptors (Bodart et al., 1996), have functional luteinizing hormone (LH)/chorionic gonadotrophin (hCG) receptors (Rao et al., 2004) and respond to activin A (activins and inhibins are related glycoproteins that modulate pituitary follicle stimulating hormone (FSH) and consequently sex steroid production) by decreased sex steroid secretion (Vanttinen et al., 2003). H295R cells are also reported to respond to tumour necrosis factor which increases steroidogenesis (Mikhaylova et al., 2007) and epidermal growth factor and prostaglandins (Watanabe et al., 2006). The general non-steroidogenic molecular biology of this cell line is also described in the literature. As such, the H295R cell line is an extremely well characterised and versatile tool for endocrine toxicology (both the assessment of adrenocortical function and the process of steroidogenesis as a whole) and it would be a good stand- ard model. As it is a human cell line it is also particularly relevant for toxicological hazard assessment and extra- polation: Oskarsson et al. (2006) have compared the H295R cell line with normal adult human adrenal over a number of endpoints and report good correlation of response, which was improved with the addition of forskolin to H295R cells, and this also supports the H295R cell line as a relevant model.
An important consideration in the use of any cell line is the existence of multi-drug resistance proteins that
act as chemical efflux pumps. The over expression of such proteins may result in the test compound being pumped out of the cell leading to insensitivity. Little is known about multi-drug resistance proteins affecting the expression of toxicity specifically in the H295R cell line, and none of the reports cited above or in Table 1 appear to have examined this, but Oskarsson et al. (2006) report good correlation of response to toxicity between the H295R cells and normal human adrenal. However, multidrug resistance genes/P-glycoprotein efflux trans- porter are known to express in both the normal human adrenal cortex (Srinivas et al., 2006) and human adreno- cortical carcinoma (Ahlman et al., 2001) and as such, this is a variable requiring consideration. Interestingly, the synthetic glucocorticoid dexamethasone has been shown to reduce doxorubicin P-glycoprotein efflux in rat hepato- cytes (Fardel et al., 1993) raising the question of whether natural adrenal corticosteroids have similar actions in other cells including cultured adrenocortical cells.
Conclusion
A strategy has been proposed to evaluate adrenocortical toxicity. The concept is to develop a standardised approach to assess the most important toxicological effect on the adrenal, which is considered to be functional adrenocortical suppression. The in vivo ACTH challenge study is designed to provide such information and is considered particularly amenable to regulatory applica- tion. The H295R human cell line is also proposed as a method to elucidate mechanisms and is recommended as a research tool. Both methods require intra- and inter- laboratory validation with known positive and nega- tive control standards and standard operating procedure development. Both techniques provide hazard assess- ment data but it will be regulatory agencies that must consider the significance of such data in risk extrapola- tion models.
Finally, chemically induced adrenal suppression, with the cases of etomidate and aminoglutethimide, has been a clearly documented example of human iatrogenic endo- crine disruption. Environmentally, sentinel species, such as fish, have also shown evidence of adrenal endocrine disruption attributed to exposure to a wide variety of chemicals (e.g. Hontela et al., 1992; Quabius et al., 1997; Norris et al., 1999; Bisson and Hontela, 2002; Harvey and Everett, 2003). The extent of human sub-clinical adrenal effects from environmental chemical exposures is unknown. Although it is unlikely that acute, low-level human environmental chemical exposures would provoke full adrenal suppression and Addisonian crisis, the extent to which environmental chemicals may act as a contribu- tory factor to human adrenal conditions following chronic low-level exposures will remain unknown unless purpose- fully studied.
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