Adrenal Toxicology PDF

Preface

Despite the adrenal gland being the most common target within the endocrine system (Ribelin, 1984), adrenal dysfunction is poorly recognized in toxicology. The adrenal is a defined vital organ with a primary role in the adaptation to stressful circumstances (indeed adrenocortical glucocorticoid production is the single most important physiological response for survival of an organism post-injury or infection, e.g., Munck et al., 1984), and toxicological pathology in regulatory studies often disregards adrenocortical histological findings as secondary to stress, which can be inappropriate without evidence that the adrenal cortex is functionally competent. For example, adrenal hypertrophy may certainly be due to stress-induced oversecretion of adrenocorticotropic hormone (ACTH), but this condition may also arise from more serious adrenocortical steroidogenic enzyme inhibition, the consequent loss of glucocorticoid (cortisol or corticosterone) secretion, and reduced or abolished feedback control of pituitary ACTH secretion. The resultant uncontrolled ACTH hypersecretion can then overstimulate the cortex to produce hypertrophy. Genuine stress-related changes encountered in toxicity studies are generally considered to be of minimal toxicological consequence, being physiologically adaptive responses that are reversible upon withdrawal of treatment. By contrast, pharmacotoxicological suppression of steroidogenesis can be a serious condition leading to Addisonian crisis (adrenal insufficiency characterized by lethargy, hemodynamic instability, and cardiovascular collapse) and death. Indeed, there are many examples of drugs and chemicals known to inhibit critical adrenocortical enzymes, potentially producing adrenal incompetence, insufficiency, or suppression, and several examples have been discovered in patients following unexpected side-effects and fatalities apparently not adequately detected, or indeed ignored as inconsequential, in preclinical toxicology.

The adrenal medulla and cortex, respectively have acute and prolonged adaptive functions in the stress response, but it is the cortex that has additional important roles in regulating water and electrolyte balance, metabolism, inflammation, immune function, and various reproductive and developmental processes depending on life stage and species. It is therefore surprising that the adrenal has been neglected in endocrine toxicology and this has been pointed out as a critical omission in a regulatory toxicology context (Harvey and Johnson, 2002; Harvey and Everett, 2003; Harvey and Everett, 2006; Hinson and Raven, 2006; Harvey, Everett, and Springall, 2007). The United States Environmental Protection Agency (USEPA), Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC), in not incorporating adrenal evaluation studies in its endocrine disruption strategy, failed to recognize the adrenal as an important endocrine gland influencing health, development, and survival fitness, or the unique vulnerability of the adrenal to toxic insult (see chap. 1) compared with other endocrine organs. Further, the adrenal cortex is also unique in possessing almost universal steroidogenic capability, and this was also overlooked in the recommendations for the development of models/assays to examine the effects of chemicals on steroidogenesis. The human adrenocortical carcinoma derived H295R cell line has been suggested as a useful system to address both issues (e.g., Harvey and Everett, 2003; Harvey, Everett, and Springall, 2007). Other regulatory bodies have since partially rectified this situation and recognized the importance of the adrenal in toxicology, or at least the utility of adrenocortical cells as a model for steroidogenesis as a whole, and the OECD has recently undertaken a program to validate the H295R cell line as a universal model to evaluate steroidogenic toxicity (e.g., Hecker et al., 2007). Oskarsson et al. (2006) report that steroidogenic gene expression following chemical challenge is comparable between the H295R cell line and the normal human adrenal demonstrating the validity of this cell line. The consensus is that the H295R cell line is currently the best available model, and even though ACTH receptors are not well expressed, effective downstream challenge augmentation can be achieved by drugs, such as forskolin.

The number of literature studies on adrenocortical toxicity has risen markedly over the past decade, which indicates the growing scientific and regulatory interest in the adrenal as a target for "endocrine disruption," with the majority of studies using in vitro techniques. Numerous laboratories are now using the H295R cell line to investigate the diverse effects of chemicals on molecular mechanisms of adrenocortical toxicity, and/or the general process of steroidogenesis, using steroid production, gene regulation, and enzyme expression as endpoints see Sanderson et al., 2001; Muller-Vieira et al., 2005; Gracia et al., 2006; Hecker et al., 2006; Oskarsson et al., 2006; Furuta et al., 2008 and Stigliano et al., 2008 for recent examples of the range of endpoints that can be assessed in this cell line, and chapters 7 and 8 for thorough reviews). While this is an important step forward, in vitro systems will not detect drugs or chemicals that affect adrenal function higher in the hypothalamo-pituitary axis, or effects on carrier proteins, and therefore there is also a need to validate in vivo models. A strategy for evaluation of drug and chemical effects on adrenocortical function has been proposed (Harvey, Everett, and Springall, 2007, and also see chap. 1 for further details) and involves a short-term in vivo adrenal challenge test in rodents (or other laboratory species if indicated—see Colagiovanni et al., 2006, and in this volume, for information on adrenal function and mechanism evaluation in the dog) coupled with the H295R in vitro assay to shed light on molecular sites of toxicity/functional inhibition.

The primary purpose of this text is to review the scope of, and developments in, adrenal toxicology. It is specifically designed to be both complementary to, and an update of, the first text in this area The Adrenal in Toxicology: Target Organ and Modulator of Toxicity (Harvey, 1996a). The present text has minimal overlap with the former, but reviews the major developments in the field over the last decade, and identifies research requirements including the validation of standardized models and methods. The focus of this new text is on the adrenal as a target organ, both in the main mammalian species used in pharmaceutical and chemical regulatory toxicology and environmental sentinel species, and also on the advances in identifying molecular mechanisms of action. Most toxicological research has focused on the cortex rather than the medulla, which reflects its wider and more complex role in physiological processes, and this is also by necessity reflected in this text, although pathology of the medulla is examined in the rodent and human.

Following an introduction and overview of adrenal endocrine control and toxicology covering the range of toxicants, targets, mechanisms, interactions, models, and species differences (see chap. 1), there is a section covering the endocrinology and pharmacology of the adrenal in health and disease. An exemplary chapter on human adrenal dysfunction (see chap. 2) reinforces the critical importance of the correct function of this gland, both medulla and cortex, and from this, the potential consequences of drug or chemical-induced dysfunction may be inferred. For example, many adrenal diseases and syndromes are generally due to faults in gene, enzyme, or receptor expression, and the knowledge derived from human medicine of the molecular basis of dysfunction can assist in identifying the significance and consequences of pharmacotoxicologically induced effects on these targets. Further, the fact that many of the enzymes involved in human adrenocortical disorders can be pharmacologically manipulated (Hakki and Bernhardt, 2006) raises the real possibility that these adrenal conditions could occur as a consequence of off-target toxicity from drugs and chemicals, both in normal subjects, but especially in patients predisposed to develop the natural etiological factors of the disease, where additional insult may accelerate or precipitate onset. In addition, this section also provides a highly authoritative review (see chap. 3) of the endocrinology, pharmacology and pathophysiology of the hypothalamopituitary- adrenal (HPA) axis, including new emerging knowledge of developmental effects of HPA hormones. The early life influence of glucocorticoid hormones is an important pharmacological field with immense potential application; adrenal glucocorticoid analogues have well documented uses to accelerate maturation of premature/fetal lung, but have also long been known to have adverse effects on general growth and development in terms of teratogenicity (Hawkins, 1983). New evidence suggests they may also be associated with adult susceptibility to major diseases such as hypertension, type 2 diabetes, coronary heart disease, hyperlipidemia and nervous system disturbance following early life exposure. The pharmacology of glucocorticoids and effects on immune and inflammatory responses is introduced (properties which have been exploited in one of the most important classes of medicines in the past 50 years, for example, in asthma and allergy— see also Harvey, 1996a, and chapters therein for accounts of the well-known classical pharmacology of adrenal glucocorticoids and their synthetic analogues).

Thus, inappropriate adrenocortical steroid secretion due to pharmacotoxicological action, either oversecretion or suppression, can have far reaching effects on organ systems and tissues throughout the body. Understanding molecular mechanisms of drug action is fundamental to both pharmacology and toxicology, especially if the toxicity is due to suprapharmacological effects, and thus the purpose of this section is to outline the importance of the adrenal and its physiology in health and disease, and thereby identify potential pharmacotoxicological targets and the consequences of their manipulation and dysfunction.

The main section of the book details mammalian adrenal toxicology in the rodent, dog, and primate, both medulla and cortex. There are two definitive reference chapters on adrenal pathology, one on toxicopathology of the rodent adrenal medulla (see chap. 4) and the other on pathology of the primate adrenal (see chap. 5), and both provide microscopic histopathological examples and expert descriptions of the conditions affecting the adrenal. There is a chapter on evaluation of HPA function in the dog and in vivo mechanism elucidation (see chap. 6), which is a unique case illustration of a strategy for investigation of the site of toxicity within the integrated endocrine axis, developed to identify a mechanism and solve a specific regulatory preclinical toxicology question. There are two stateof- the-art chapters on in vitro mechanistic approaches from leading researchers in the field, one covering a comparison of the strengths and weaknesses of the various mammalian and human cell lines available (see chap. 8), and the second focusing exclusively on the use of the human H295R cell line (see chap. 7). This cell line is becoming the standard research system for toxicological evaluation of both adrenocortical function and the process of steroidogenesis as a whole (discussed earlier) and, having the advantages of being derived from human tissue and retaining complete steroidogenic functionality, this cell line can be used in mechanistic research with in vivo models to evaluate the majority, if not all, of the theoretically possible biochemical mechanisms of toxicity. The final chapter in this section is an extensive review of pharmacotoxicological interactions of adrenal hormones and the toxic response at the cellular and molecular levels (compare this with Harvey, 1996b, which discussed such interactions at the target organ and whole body level), detailing the molecular mechanisms influencing these effects including regulation of genes, cell cycle pathways and apoptosis (see chap. 9). Also discussed are the interesting and complex circumstances where these responses may be either beneficial or detrimental depending on the toxic insult and a variety of other interactive variables and conditions (see also chap. 1 on how coincidental physical stress as an interactive variable at the whole body level uncovered occult subclinical enzyme inhibition and severe adrenal suppression in patients treated with etomidate). Essentially, this section is designed to cover the major principles of adrenal toxicology at the tissue, cellular, and molecular levels; reinforce the complexity of adrenocortical hormone actions and interactions; and provide a primary reference source of examples, strategies, and methods for toxicologists and pathologists in both regulatory and research fields.

The final section covers adrenal dysfunction in environmental species and draws on field leaders who have documented adverse effects in fish (see chap. 10) and birds (see chap. 11) from insidious ambient environmental exposures to chemical pollutants. Such species can be considered as sentinel or indicator species, and the fact that real environmental exposures have been shown to cause adrenal endocrine disruption in wildlife prompts the question of whether human adrenal function could also be compromised by low-level chemical exposures, producing occult subclinical effects. In such a case, further adrenal insult may combine with any preexisting functional deficiency to have interactive, additive, or disproportionally pronounced effects (especially in sensitive or predisposed individuals as discussed earlier), or indeed to overtly precipitate a toxicological reaction comprising an adrenal insufficiency crisis (although there is currently no evidence of adrenal suppression in humans from environmental chemical exposure, this is mainly because it has not been studied). If continuous or combined low-level exposures are indeed influencing adrenal function in a variety of wildlife species, this could ultimately affect survival fitness especially in adverse environmental circumstances. While endocrine disruption involving estrogenic/antiandrogenic effects often produces gross macroscopic morphological evidence in target/sentinel species, for example, to reproductive organs, this is generally not the case with adrenal dysfunction, and indeed the ultimate endpoint of adrenal dysfunction is death without obvious cause if there has been rapid onset of contributory factors (and as detailed above, these may be complex interactions where no single factor is an obvious cause). As with adrenal toxicity evaluation in mammalian regulatory toxicology, the challenge in environmental or ecotoxicology is to find reliable and accurate methods of identifying effects, made more difficult with wildlife species requiring minimal disturbance in situ. This section therefore extends the scope of adrenal toxicology into the field of "environmental endocrine disruption," and although the specific examples of fish and birds are of primary interest to the ecotoxicologist, humans are also environmentally exposed to combinations of a variety of chemical pollutants, and the toxicological hazards and potential consequences to human health can be at least recognized in principle (for example, see Furuta et al., 2008, on the effects of diesel exhaust chemicals on adrenocortical cell function in vitro). From this, there is a research need for properly controlled studies into human exposures to known adrenal toxicants, and for potential effects to be evaluated from which risk assessments can be developed. Human studies are notably lacking in the "endocrine disruption" literature in general, and addressing this should be considered a research priority.

Adrenal dysfunction can lead to significant morbidity and mortality across species. This has been well documented for humans in the medical literature, and even though drugs such as the anesthetic agent etomidate have been known for more than 20 years to inhibit critical enzymes on the cortisol pathway as an off-target side effect, new cases of etomidate-induced Addisonian crisis continue to be described, even after a single dose and as new variables come to light (Lundy et al., 2007). There is clearly a need to develop an assessment strategy to assess potential effects on adrenal function, both in mammalian regulatory toxicology programs of drugs and chemicals for human risk extrapolation, and also in environmental toxicology to assess the potential effects on wildlife and humans from low-level chemical exposures. In regulatory toxicology of drugs and chemicals, Harvey, Everett, and Springall (2007) have suggested that a rodent in vivo adrenal function test is developed where, for example, rats treated with a test compound are also challenged withACTH, and corticosterone secretion is assessed as an indicator of adrenal competence (see also chap. 1). This in vivo ACTH challenge test could also be applied to cortisol secretion in the dog (see chap. 6) or the primate to assess adrenal function in these species, if indicated by toxicological findings. An in vitro test using H295R cells and cortisol secretion and enzyme expression analyses was also suggested to examine and characterize mechanisms of toxicity (Harvey and Everett, 2003; Harvey, Everett, and Springall, 2007). This cell line is currently undergoing validation under OECD protocols as a method of examining the effects of chemicals on steroidogenesis (Hecker et al., 2007), but at present this appears to only involve sex steroid production. It would be prudent to extend this validation to include glucocorticoid and mineralocorticoid secretion with appropriate positive- and negative-control compounds. There is a similar need to develop and validate laboratory ecotoxicology tests in representative and ecologically important species such as fish. Thus, while the primary purpose of this text has been to highlight the importance of the adrenal gland in health and disease and review the scope and developments in adrenal toxicology, another major goal is to call for a strategy of standardized adrenal toxicology methodology applicable to regulatory toxicology, for the initiation and validation of such protocols in both mammalian and environmental toxicology, and for research into human exposures and assessment of their potential effects.

Finally, this text is designed for toxicologists and pathologists working in research and regulatory fields and pharmaceutical and chemical industry laboratories, requiring a reference source in an area rapidly gaining prominence in a regulatory context, namely adrenal toxicology and adrenal endocrine disruption. It will also be of interest to those working in in vitro, mechanistic and molecular toxicology, and to pharmacologists, environmental health scientists, ecotoxicologists, risk assessors, and endocrinologists.