Adrenal Gland: Structure, Function, and Mechanisms ...

5 downloads 0 Views 224KB Size Report
Antlers Adams Mark Hotel. Colorado Springs, CO. Non-Rodent Species in Toxicologic Pathology. 2003. June 16–19. Westin Savannah Harbor Resort.
Toxicologic Pathology http://tpx.sagepub.com/

Adrenal Gland: Structure, Function, and Mechanisms of Toxicity Thomas J. Rosol, John T. Yarrington, John Latendresse and Charles C. Capen Toxicol Pathol 2001 29: 41 DOI: 10.1080/019262301301418847 The online version of this article can be found at: http://tpx.sagepub.com/content/29/1/41

Published by: http://www.sagepublications.com

On behalf of:

Society of Toxicologic Pathology

Additional services and information for Toxicologic Pathology can be found at: Email Alerts: http://tpx.sagepub.com/cgi/alerts Subscriptions: http://tpx.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://tpx.sagepub.com/content/29/1/41.refs.html

Downloaded from tpx.sagepub.com by guest on May 24, 2011

T OXICOLOGIC P ATHOLOGY, vol 29, no 1, pp 41 – 48, 2001 Copyright 2001 by the Society of Toxicologic Pathologists

Adrenal Gland: Structure, Function, and M echanisms of Toxicity T HOMAS J. R OSOL ,1 J OHN T. Y ARRINGTO N , 2 J OHN L ATENDRES SE, 3 2

AND

C HARLES C. C APEN 1

1 The Ohio State University, Columbus, Ohio 43210 Wil Research Laboratories, Inc., Ashland, Ohio, and 3 Pathology Associates, Inc., Jefferson, Arkansas

A BSTRACT The adrenal gland is one of the most comm on endocrine organs affected by chemically induced lesions. In the adrenal cortex, lesions are more frequent in the zona fasciculata and reticularis than in the zona glomerulosa. The adrenal cortex produces steroid hormones with a 17-carbon nucleus following a series of hydroxyla tion reactions that occur in the m itochondria and endoplasm ic reticulum. Toxic agents for the adrenal cortex include short-chain aliphatic compound s, lipidosis inducers , amphiphilic compounds , natural and synthetic steroids, and chemicals that affect hydroxyla tion. Morphologic evaluation of cortical lesions provides insight into the sites of inhibition of steroidogenesis. The adrenal cortex response to injury is varied. Degeneratio n (vacuolar and granular), necrosis, and hem orrhage are common Ž ndings of acute injury. In contrast, chronic reparative processes are typically atrophy, Ž brosis, and nodular hyperplasia. Chem ically induced proliferative lesions are uncom m on in the adrena l cortex. The adrenal medulla contains chrom afŽ n cells (that produce epinephrine, norepineph rine, chromogranin, and neuropeptides) and ganglion cells. Proliferative lesions of the medulla are common in the rat and include diffuse or nodular hyperplasia and benign and malignant pheochrom ocytoma. Mechanism s of chromaf Ž n cell proliferation in rats include excess growth hormone or prolactin, stimulation of cholinergic nerves, and diet-induced hypercalcem ia. There often are species speciŽ city and age dependenc e in the developme nt of chemically induced adrenal lesions that should be considered when interpreting toxicity data. Keywords.

Adrenal cortex; adrenal medulla; corticosteroid biosynthesis; chromafŽ n cell; degeneration; toxicolog y

prominent capillaries. The cells are polyhedral and have many intracellular lipid droplets. This zone produces glucocorticoids. The zona reticularis is also composed of polyhedral cells, whose arrangement is less linear and more as round nests or clumps of cells. The zona reticularis produces glucocorticoids and in som e species small amounts of sex steroids, namely, androgens, estrogens, and progestins. This zone is more distinct in rats compared to m ice.

I NTRODUCTION The adrenal gland is reported to be the m ost com mon endocrine organ associated with chemically induced lesions (17). It is especially important to understand the structure and function of the adrenal gland to correctly interpret the signiŽ cance and mechanisms of drug-induced lesions. The adrenal cortex is required for life, particularly the secretion of aldosterone, but the functions of the medulla are not essential for life. Current reviews on the toxicity of the adrenal gland have been prepared by Capen et al (1), Colby and Longhurst (3), Hinson and Raven (8), Tishler (19), Woodm an (25), Yarrington and Reindel (29), and Yarrington et al (27).

B LOOD S UPPLY

OF THE

A DRENAL G LAND

There are unique anatomical features of the adrenal gland blood supply that are important for its function and development of lesions. The gland is supplied by arterioles that penetrate the capsule, lose their m uscular wall, and form a capillary bed that supplies the adrenal cortex. The arterioles that penetrate the capsule have a rapid reduction in lumen diam eter and are a common site of embolization of bacteria or tumor cells. The capillaries of the cortex supply blood to the m edulla via the corticalmedullar y portal blood system. This results in a high concentration of glucocorticoids in the blood supplied to the medulla. Some of the arterioles directly supply the medulla with blood, so the medulla has 2 sources of blood, namely, cortical capillaries and primary arterioles. Blood leaves the adrenal gland via medullary veins.

N ORMAL S TRUCTURE OF THE A DRENAL C ORTEX The adrenal cortex is composed of 3 distinct zones. The outer zone is the zona glomerulosa and is composed of a thin region of colum nar cells arranged in an arched or arcuate pattern. This zone is also called the zona multiform is in animals because of its different patterns of arrangement of secretor y cells. The zona glomerulosa produces the steroid hormone aldosterone, which is responsible for increasing sodium reabsorption and stimulating potassium excretion by the kidneys and thereby indirectly regulating extracellular  uid volume. Loss of this zone or the inability to secrete aldosterone may result in death due to retention of high levels of potassium with excess loss of sodium, chloride, and water. The zona fasciculata is the thickest zone ( 70% of the cortex) and is composed of columns of secretor y cells separated by

U LTRASTRUCTURE

OF THE

A DRENAL C ORTEX

Ultrastructural features of the cortical cells can be ver y useful for their identiŽ cation. All 3 zones have prominent mitochondria that can be distinguished by the shape of their cristae (Table 1). Cells of the zona glomerulosa are characterized by prom inent mitochondria and Golgi ap-

Address correspond ence to: Dr Thomas J. Rosol, The Ohio State University, Department of Veterinary Biosciences, 1925 Coffey Road, Columbus, OH 43210; e-mail: rosol.1@ osu.edu.

41 Downloaded from tpx.sagepub.com by guest on May 24, 2011

0192-6233 /01$3.00 $0.00

42

ROSOL ET AL

T OXICOLOGIC P ATHOLOGY

T ABLE 1.—Ultrastructural features of the adrenal cortex.

Lipid droplets Mitochondria Golgi apparatus Smooth endoplas mic reticulum Lysosom es

Zona glomerulosa

Zona fasciculata

Zona reticularis

(Lea ike cristae)

(Vesicular cristae)

(Tubulovesicula r cristae)

paratus, whereas cells of the zona fasciculata have many cytoplasmic lipid droplets, mitochondria with vesicular cristae, and abundant smooth endoplasmic reticulum . The zona reticularis is distinguished by the presence of prom inent lysosomes in the cytoplasm. F ETAL A DRENAL G LAND A specialized fetal adrenal cortex exists in primates during late gestation (13). The cortex is composed of large polyhedral cells that produce abundant cortisol and estrogen precursors. The hormones secreted by the cortex are important for norm al development of the fetus, and the steroid precursor dihydroepiandrosterone is converted to estrogen by the placenta. The cells of the fetal cortex are produced in the outer cortex and m igrate medially, where they undergo hypertrophy and eventually apoptosis. After birth, there is rapid regression, apoptosis, and lysis of the fetal cortex with dilatation of cortical capillaries and replacement by the typical 3 cortical zones. It is important not to misinterpret this as a lesion in neonatal primates since it represents physiological replacem ent of the fetal cortex with the deŽ nitive postnatal adrenal cortex. X -Z ONE IN M ICE The X-zone in the mouse adrenal cortex is a similar unique physiologic phenomenon as the fetal cortex in primates. In contrast to the fetal cortex of primates, the X zone develops postnatally in the inner cortex of mice and is fully formed at weaning. Its function is unknown, but it m ay be sim ilar to the fetal zone in primates. After weaning, the X -zone degenerates at variable rates, depending on the sex of the mouse. In male mice, the X zone undergoes degeneration at puberty with accumulation of intracellular fat globules. In unbred females, the zone undergoes slow regression and degeneration. In pregnant females, it undergoes vacuolar degeneration during the Ž rst pregnancy. As with the fetal zone in primates, it is important not to misinterpret the degeneration associated with regression of the X -zone in m ice as a lesion. S PONTANEOUS L ESIONS OF THE A DRENAL C ORTEX There are a variety of spontaneous lesions in the adrenal cortex that m ust be differentiated from chemically induced lesions (5). Amyloidosis. Both prim ary and secondary amyloidosis occurs in aged m ice (18). Primary amyloidosis is associated with certain strains of mice, whereas secondary amyloidosis is associated with chronic in amm ation, such as seen in mice treated with vaccine adjuvants.

Ceroid and lipofuscin deposition. Aged mice and rats may develop ceroid deposition (‘‘brown degeneration’’) in adrenal cortical cells and m acrophages in the inner zone (4, 16). Vacuolar degeneration. Some animals, particularly cats and rats, may develop spontaneous vacuolar degeneration of adrenal cortical cells in the zona fasciculata. This lesion can m imic vacuolar degeneration due to chemically induced toxicity. If the vacuolar degeneration is severe, there will be loss of cortical cells, possibly mineralization, and vascular ectasia. Telangiectasis. Both aged rats and mice develop telangiectasis in the adrenal cortex because of marked dilatation of cortical capillaries after loss of parenchymal cells. Extramedullary hematopoiesis. Extram edullary hematopoiesis is occasionally observed in the adrenal cortex and may contain erythrocytic and/or granulocytic cells. This change must be differentiated from in ammation. Spindle cell hyperplasia. The adrenal cortex contains both polyhedral cells and spindle cells. The function of the spindle cells is unknown, but they appear to represent a morphological variant of the epithelial cells in a subcapsular location of the cortex. Both mice and ferrets can develop spindle cell hyperplasia or neoplasia (6). The spindle cells in ferrets are associated with production of estrogen interm ediates. Spindle cell hyperplasia is most com mon in the subcapsular region in mice and is enhanced after gonadectomy (7). S TEROID H ORMONE B IOSYNTHESIS IN THE A DRENAL C ORTEX The adrenal cortex is responsible for production of both mineralocorticoids and glucocorticoids. The precursor of steroid horm ones is cholesterol, which has the 17carbon steroid nucleus. Cholesterol is converted to steroid horm one interm ediates and mature horm ones by cytochrom e P -450 enzym es in the m itochondria and smooth endoplasmic reticulum. Synthesis begins in the mitochondria, continues in the endoplasmic reticulum , and is com pleted in the m itochondria. Therefore, shuttling of steroid horm one precursors between the 2 cytoplasmic com partm ents is important in the multiple steps of hormone synthesis. Secretion of steroid horm ones is immediate because of their lipid solubility, and there is a lack of storage of preformed hormone in the cortical cells. Therefore, blood concentrations re ect rates of synthesis of hormone. Secretion of the steroid hormones follows a circadian rhythm. In nocturnal animals (eg, rats, mice, and cats), secretion is greatest in the early evening. In daytim e an-

Downloaded from tpx.sagepub.com by guest on May 24, 2011

Vol. 29, No. 1, 2001

ADRENAL GLAND TOXICITY

43

F IGURE 1. Steroidogenesis in the adrenal cortex. Enzymatic conversion of cholesterol to pregnenalon e by CYP11A1 is the rate-limiting step for synthesis of the glucocorticoids . ACAT, acyl coenzym e A: cholesterol acyltransferase; ACTH, adrenocorticoto pic hormone; CYP11A1, 20 , 22Rhydroxylas e cholesterol side-chain cleavage; CYP11B1, 11 -hydroxylase and 18-hydroxylas e activities; CYP11B2, aldosterone synthase; CYP17, 17 hydroxylas e and 17 – 20 lyase activities; CYP21, 21-hydroxylase ; HDL, high-density lipoproteins; LDL, low-density lipoproteins; nCEH, neutral cholesterol ester hydrolase.

imals (eg, dogs and primates), secretion is greatest in the morning. Most anim als have a m ild decrease in secretion with age. The steroid horm ones are bound in serum to corticosteroid-binding globulin (transcortin), which increases the circulating half-life and solubility of the hormones. Unbound steroid is free to interact with target cells either to exert metabolic effects or to be transformed into an inactive metabolite. The corticosteroids are metabolized in the liver by hydroxy lation and conjugation reactions and excreted in the bile. Cholesterol, the precursor for corticosteroid horm one synthesis, can be derived from multiple sources (9). Cholesterol can be synthesized de novo in cortical cells from acetate or can be absorbed as low - or high-density lipoproteins (HDLs) (Figure 1). Only HDL uptake occurs in rats. Cholesterol is stored as cholesterol acetate in neutral lipid droplets, which serves as a pool of readily available cholesterol for corticosteroid biosynthesis. The Ž rst steroid hormone produced by cortical cells from cholesterol is pregnenolone by the action of mitochondrial cytochrom e P-450 (CYP) 11A1 (20 , 22R -hy droxylase cholesterol side-chain cleavage). This reaction

is important since it is the rate-limiting step of steroid hormone biosynthesis (Figure 1). This step is under the control of ACTH secreted by the pituitary gland. ACTH binds to cell membrane receptors linked to G -proteins and stim ulates increased cytoplasmic cAMP and increased availability of cholesterol to CYP11A1, which results in increased pregnenolone synthesis. After synthesis of pregnenolone in rodents, synthesis of glucocorticoids continues in the m itochondria and the smooth endoplasmic reticulum to form corticosterone. This is the principal glucocorticoid in rats, mice, rabbits, birds, reptiles, and amphibians. In other species, such as ham sters, dogs, cats, nonhuman primates, hum ans, and Ž sh (teleosts), the smooth endoplasmic reticulum contains additional hydroxylases that are responsible for synthesis of cortisol (Figure 1). Cortisol is produced in greater amounts compared to corticosterone in these species and represents approximately 80% of the glucocorticoid production. In addition, androgens may be produced by the cortical cells, especially in the zona reticularis. The androgens produced by the zona reticularis can be metabolized to testosterone or estrogens by the cortical cells

Downloaded from tpx.sagepub.com by guest on May 24, 2011

44

ROSOL ET AL

themselves or by metabolic pathways in other organs, such as the gonads. Species that produce predom inantly corticosterone (such as rats and mice) have little sex hormone production by the adrenal glands. Aldosterone is the principal mineralocorticoid produced in the zona glomerulosa since CYP11B2 is found only in this zone. Angiotensin II and potassium stim ulate aldosterone production. Angiotensin II is produced from a horm one precursor, angiotensinogen (produced in the liver), by the action of the enzym e renin, produced by the juxtaglom erular apparatus of the kidney. Angiotensinogen is Ž rst cleaved to a decapeptide, angiotensin I, by renin and subsequently to angiotensinogen II by an angiotensin-converting enzyme (ACE) present in several tissues. Angiotensin II acts as a trophic hormone to increase aldosterone production, which acts on target cells in the kidney to conser ve sodium , excrete potassium, and increase blood volum e. M ECHANISMS OF T OXICITY OF THE A DRENAL C ORTEX Adrenal cortical cells contain large stores of lipid used as substrate for steroidogenesis. M any com pounds that are toxic for the adrenal cortex are lipophilic and accumulate in these lipid-rich cells. Impaired steroidogenesis. Impaired steroidogenesis is an important mechanism of toxicity in the adrenal cortex. It can occur by inhibition of cholesterol biosynthesis or metabolism and by disruption of cytochrome P-450 enzym es. Both of these mechanism s will lead to the accumulation of increased cytoplasmic lipid in the form of discrete droplets. Toxin activation by CYP-450 enzymes. Toxins may be activated by many of the cytochrome P-450 enzymes in the cortical cells. Activation of toxins can result in the generation of reactive oxygen m etabolites, result in m embrane damage, and produce phospholipidosis in the cells. Exogenous steroids. Exogenous steroids can disrupt norm al function and structure of the adrenal cortex. Exogenous agonists will induce negative feedback inhibition of ACTH secretion by the pituitary and will result in atrophy of the zona fasciculata and reticularis. Som e steroids, such as the sex steroids, can induce proliferative lesions in the adrenal cortex. Exogenous steroid antagonists will block steroid hormone action, lead to increased ACTH secretion, and diffuse hyperplasia of the cortex. DNA damage. Agents that dam age DNA, such as carcinogens or radiation, may induce neoplasia of the adrenal cortex. There is considerable species variation in the response of the adrenal cortex to exogenous chemicals. This is due to both inherent differences in the sensitivity to certain drugs and differences in the metabolic pathways of steroidogenesis. An interesting exam ple is o,p -DDD. o,p DDD (M itotane) was originally developed to treat metastatic adrenal cortical cancer in humans; however, humans are relatively insensitive to the effects of o,p -DDD, and the drug was not useful in the treatment of adrenal cancer. In contrast, dogs are more sensitive to the effects of o,p -DDD, and it has been used effectively to treat pituitary-dependent hyperadrenocorticism due to autonom ous secretion of ACTH by pituitary (corticotroph) tu-

T OXICOLOGIC P ATHOLOGY

mors in a dose-dependent m anner. o,p -DDD is a selective toxin for the zona fasciculata and reticularis, thereby sparing the important functions of the zona glomerulosa. A SSESSMENT OF F UNCTION OF THE A DRENAL C ORTEX It m ay be useful to measure the function of the adrenal cortex. This can be accom plished by measuring glucocorticoid horm one concentrations in the blood or urine (expressed as a ratio to creatinine). It is important to remember the diurnal variations in secretion. Provocative testing is a useful tool to evaluate the functional capacity of the zona fasciculata and reticularis by measuring the increase in secretion of glucocorticoids in response to exogenous ACTH. Light and electron microscopy and histomorphom etr y (cortico:medullary ratio and width of different cortical zones) are useful to characterize lesions that may disrupt the function of the cortical cells. M ANIFESTATIONS OF T OXICITY OF THE A DRENAL C ORTEX Acute toxicity. Acute toxicity of the adrenal cortex can have multiple morphologic manifestations. Impaired steroidogenesis is a comm on toxin-induced change that can result in excess steroid precursors and cytoplasmic vacuolation. Early the cytoplasmic vacuoles are small, but then they coalesce to form larger vacuoles. Triar yl phosphates are an example of a group of organophosphates that result in impaired cholesterol metabolism and increased cytoplasmic lipid vacuoles (10). Usually the vacuolation begins in the inner cortex and progresses to the outer zones. If the vacuoles are clear by light microscopy, they usually represent lipid, whereas granular degeneration often represents swelling and damage to mitochondria. DMNM ( -[ 1,4-dioxido-3-methylquinoxalin-2-yl ] N-methylnitrone) is an antibiotic that causes impaired steroidogenesis, likely by blocking the conversion of cholesterol to pregnenolone. After acute exposure, it results in cytoplasmic vacuolation of the zona fasciculata and reticularis (26, 28). Chemicals that induce severe vacuolar degeneration, like DMNM, often lead to loss of adrenocortical cells due to necrosis, cell lysis, or apoptosis. Chemicals such as the DDT derivative, o,p -DDD, which dam age mitochondria, can lead to severe, acute necrosis and hem orrhage of the adrenal cortex (27). Ultrastructurally, the smooth endoplasmic reticulum and m itochondria will be swollen with disruption of the cristae. In addition, there will be an accumulation of lipid bodies due to impaired steroidogenesis. Ultrastructural evaluation of the adrenal cortex with degeneration can provide important m echanistic clues to the pathogenesis of the toxicity. This is because many of the enzym es affected by toxic chem icals are organelle speciŽ c, such as 11 -hydroxy lase (CYP11B 1) in mitochondria and the 17 - and 21-hydroxylases (CYP 17 and 21) in the SER. In addition, electron microscopy of the adrenal cortex can be used to identify lesions restricted to endothelial cells; speciŽ c organelles of parenchymal cells, such as m itochondria (DMNM , o,p -DDD, and am phenone) and SER (triparanol); and the cytoplasm of parenchym al cells, such as lipid aggregation (aniline) or phospholipid aggregation (chlorophenterm ine). Chronic toxicity. Chronic toxicity of the adrenal cortex

Downloaded from tpx.sagepub.com by guest on May 24, 2011

Vol. 29, No. 1, 2001

ADRENAL GLAND TOXICITY

can lead to atrophy, nodular regeneration, Ž brosis, or primary proliferation of cortical cells. As an example, DMNM induces atrophy of the cortex and secondary nodular regeneration. Hypertrophy of adrenocortical cells can occur if the xenobiotic chem ical results in increased circulating ACTH levels. Hyperplasia and neoplasia. Hyperplasia of the adrenal cortex is a relatively uncom mon m anifestation of chem ically induced lesions (1, 15). Diffuse hyperplasia is seen in conditions that result in excessive secretion of ACTH, such as corticotroph adenomas of the pituitary gland. Usually chemically induced cortical cell proliferation is nodular in pattern. However, nodular hyperplasia of cortical cells must be differentiated from spontaneous nodular hyperplasia in animals that are predisposed to this lesion, such as dogs and certain strains of rats. Adenoma and carcinom a uncomm only result from chemical-induced adrenal cortical toxicity. Age-related proliferative lesions in rodents are uncomm on but must be differentiated from chemically induced lesions. M ice may develop spindle cell hyperplasia (type A cells) in the subcapsular region of the adrenal cortex (7) or hyperplasia of polyhedral cells (type B cells) of the cortex, which are characteristically vacuolated. Spontaneous adenomas are uncom mon, but ovariectom ized mice or mice of the NH strain whose ovaries stop functioning early in life have an increased incidence of adenomas, likely because of the stimulatory effects of pituitary gonadotropins. Spontaneous carcinom as are rare. Phospholipidosis. Phospholipidosis of cortical cells results from 2 m echanisms. Certain toxins are activated by the cytochrome P -450 enzymes to form reactive oxygen species, resulting in dam age to microsomal and m itochondrial membranes. In addition, amphophilic com pounds can intercalate in m embranes and disrupt norm al membrane turnover. The ultrastructural lesions of phospholipidosis are characterized by cytoplasmic inclusions and lysosomal bodies com posed of whorls of membranes and condensed cytoplasmic com ponents. C LASSES OF T OXINS OF THE A DRENAL C ORTEX Different classes of chemicals can lead to toxicity of the adrenal cortex by m ultiple m echanisms (1, 27). Aliphatic compounds often lead to necrosis. Lipidosis inducers that block norm al cholesterol metabolism lead to accum ulation of fat vacuoles in the cytoplasm. Amphophilic com pounds induce phospholipidosis by disturbing membrane function and turnover. Natural and synthetic estrogens and androgens can produce primary proliferative lesions in the adrenal cortex. Other chem icals disrupt oxidative enzyme reactions in the m itochondria or smooth endoplasm ic reticulum . In som e cases these effects can be speciŽ c for the adrenal gland, such as toxicity induced by o,p -DDD or DMNM. The effects are nonspeciŽ c in other cases, and multiple organs are affected, including the adrenal cortex, such as with the toxicity associated with carbon tetrachloride or cadmium. Steroid agonists inhibit secretion of ACTH by the pituitary gland and will result in atrophy of the zona fasciculata and reticularis. Steroid hormone antagonists will block the peripheral actions of the horm ones, in-

45

cluding their negative feedback mechanisms, and will result in hyperplasia of the adrenal cortex. S ELECTED E XAMPLES OF A DRENAL G LAND T OXINS AND T HEIR M ECHANISMS Triaryl phosphates. Triaryl phosphates are organophosphates used in plastics, hydraulic  uids, and lubricants that block the action of neutral cholesterol ester hydrolase with minimal inhibition of acyl coenzyme A: cholesterol acyltransferase (9, 11). This results in an accumulation of cholesterol esters in the fat vacuoles in all layers of the adrenal cortex. Interestingly, corticosterone concentrations are norm al since synthesis of cholesterol is not disrupted, thereby providing adequate cholesterol to serve as a precursor for steroid hormone synthesis. Captopril. Captopril is a drug that inhibits the peripheral actions of angiotensin-converting enzyme (ACE). This results in inhibition of the synthesis of angiotensin II from angiotensin I derived from hepatic angiotensinogen. Angiotensin II is an important stimulator of CYP11B2 and form ation of aldosterone. Reduction of aldosterone leads to increased sodium and water excretion, decreased excretion of potassium, and a reduction in blood pressure. Toxic effects of captopril include trophic atrophy of the zona glom erulosa due to reduced stimulation by angiotensin II. Carbon tetrachloride. Carbon tetrachloride is a nonspeciŽ c toxin of the adrenal cortex and results in inhibition of m icrosomal enzym es in the smooth endoplasmic reticulum. These include the 17- and 21-hydroxy lases in cortical cells. Lesions include swelling and disruption of the SER and eventual necrosis of the cells. Corticosteroid toxicity. Administration of excess glucocorticoids leads to increased negative feedback in the pituitary gland and a reduction in ACTH secretion. This will inhibit the action of CYP11A1 and lead to reduced cholesterol metabolism , accumulation of fat vacuoles in the cortical cells, and eventual atrophy of the zona fasciculata and reticularis since ACTH is important for maintaining cell num bers in the adrenal cortex. A DRENAL M EDULLA In general, the adrenal m edulla is a less common site of chemically induced degenerative lesions. However, proliferative lesions of the medulla in rats occur frequently and can be spontaneous (age related) or can result from dietary m odiŽ cations and the administration of exogenous chemicals. Proliferative lesions develop more frequently in rats than in m ice. N ORMAL S TRUCTURE

AND F UNCTION OF THE M EDULLA

A DRENAL

The cells of the adrenal medulla are derived from the neural crest in contrast to the mesodermal origin of the adrenal cortex. The secretory cells of the adrenal medulla are called chromafŽ n cells because of the form ation of colored polymers of catecholamines after exposure to oxidizing agents, such as chrom ate. They secrete epinephrine or norepinephrine into the blood in response to acetylcholine or calcium ion. There are 3 types of adrenal medullar y cells in adults (1): epinephrine cells (66 – 75% ),

Downloaded from tpx.sagepub.com by guest on May 24, 2011

46

ROSOL ET AL

T OXICOLOGIC P ATHOLOGY

soidal blood from the adrenal cortex. In the rat and mouse, norepinephrine and epinephrine are stored in separate cell types that can be distinguished ultrastructurally. The hormone-containing core of secretory granules in norepinephrine cells is electron dense and surrounded by a wide submembranous space, whereas epinephrine-containing granules are less dense with Ž nely granular m atrices. In imm ature rat adrenals, granules of varying densities m ay be found in the same cell types. The normal adult male Wistar rat adrenal contains an average of 29 nm ol norepinephrine and 71 nmol epinephrine. Pheochrom ocytomas in rats contain sparse numbers of secretory granules and produce predominantly norepinephrine, whereas most normal chrom afŽ n cells in rats produce epinephrine (20, 23). Human adrenal medullary cells contain both norepinephrine and epinephrine within a single cell. During adult life, stresses, such as hypoglycemia or reserpine-induced depletion of catecholamines, produce a re ex increase in splanchnic ner ve discharge, resulting both in catecholamine secretion and in transsynaptic induction of catecholamine biosynthetic enzym es, including tyrosine hydroxylase. Other environmental in uences, including growth factors, extracellular matrix, and a variety of hormonal signals that generate cyclic AM P, also m ay regulate the function of chrom afŽ n cells.

F IGURE 2. Synthesis of norepineph rine and epinephrine in chromafŽ n cells of the adrenal medulla. Tyrosine hydroxyla se is the rate-limiting enzyme. Enzymatic conversio n of tyrosine to DOPA and dopamine occurs in the cytoplasm . Synthesis of norepineph rine occurs in the secretory granules, which is the site of dopam ine -hydroxyla se. Norepinephrine leaves the secretory granules and is converted to epinephrine in the cytoplasm by PNMT and then re-enters the secretory granules.

norepinephrine cells (25 – 33% ), and small granule-containing cells (SGC, 4% in mice and 1% in rats). The medullary cells produce other peptides in addition to epinephrine and norepinephrine, such as met-enkephalin, substance P, neurotensin, neuropeptide Y, and chrom ogranin A. In addition, the adrenal m edulla contains presynaptic sympathetic ganglion cells. The Ž rst step in the synthesis of epinephrine is the enzym atic conversion of tyrosine to dihydroxyphenylalanine (DOPA) by tyrosine hydroxy lase (Figure 2). This is the rate-lim iting step of hormone synthesis. DOPA is converted by aromatic L -amino acid decarboxylase to dopam ine. DOPA and dopamine synthesis occurs within the cytosol. Dopam ine then enters the chrom afŽ n granule, where it is converted to norepinephrine. The norepinephrine leaves the granule to be converted into epinephrine in the cytosol by phenylethanolamine-N-methyltransferase (PNM T), and epinephrine re-enters the granule for storage in the cell. The activity of PNM T is induced by the high local concentration of glucocorticoids in sinu-

I MMUNOHISTOCHEMISTRY OF C HROMAFFIN C ELLS Imm unohistochem istry provides a new approach for the identiŽ cation of chromafŽ n cells (23). Antibodies are available that perm it epinephrine- and norepinephrineproducing cells to be distinguished even in routinely Ž xed and embedded tissue samples using antibodies to the catecholamine biosynthetic enzym es (19). Phenylethanolam ine-N-methyltransferase will be present only in the epinephrine-containing cells of the adrenal m edulla. Antibodies to chromogranin-A can be used for the dem onstration of this protein in chrom afŽ n cells, but this will also stain other types of neuroendocrine cells. C HEMICALLY I NDUCED M EDULLARY T OXICITY Medullar y chromafŽ n cells in rats are susceptible to acute necrosis and cytolysis by salinomycin, which occurs in less than 10 hours (2). However, the adrenal medulla is capable of replenishing the chromafŽ n cells in as little as 24 hours by an unknown m echanism. It appears that the adrenal medulla recruits undifferentiated chromafŽ n cells to repopulate the medulla by a rapid and orderly process of differentiation. Repopulation is not dependent on mitosis. Therefore, chem ically induced acute necrosis of the adrenal m edulla may be easily overlooked if short experim ental tim e points ( 24 hours) were not examined. P HEOCHROMOCYTOMAS Proliferative lesions of the m edulla, particularly in the rat, develop as a result of a variety of different mechanisms (1). It is important and challenging to differentiate between spontaneous and xenobiotic induced pheochromocytomas due to the relatively high incidence of these tumors, especially in male rats of certain strains. There

Downloaded from tpx.sagepub.com by guest on May 24, 2011

Vol. 29, No. 1, 2001

ADRENAL GLAND TOXICITY

is a strong genetic component to the pathogenesis of pheochromocytom as in rats, exempliŽ ed by the different incidence of pheochrom ocytomas between strains of rats. Studies from the NTP historical database of 2-year-old F344 rats have reported that the incidence of pheochromocytom as was 17.0 and 3.5% for males and females, respectively. Other strains of rats with high incidences of pheochromocytom a include Wistar, NEDH (New England Deaconess Hospital), Long-Evans, and SpragueDawley. Pheochrom ocytomas are considerably less com mon in Osborne-M endel, Charles River, Holtzm an, and WAG/Rij rats. M ost studies have reported a higher incidence in males than in fem ales. Cross breeding of animals with high and low frequencies of adrenal medullary proliferative lesions results in F 1 animals with an intermediate tumor frequency. The mean tumor size increases progressively with age, as does the frequency of bilateral and multicentric occurrence. Both spontaneous and xenobiotic induced pheochrom ocytom as are less com mon in the mouse (22). There are multiple factors in the pathogenesis of pheochromocytomas in rats. These include the genetic background, chronic high levels of growth hormone or prolactin associated with pituitary tumors, dietary factors, and stimulation of the autonomic nervous system . Chronic reserpine administration results in decreased blood pressure, neural stimulation of the adrenal medulla, and development of hyperplasia and pheochromocytoma in the rat (24). In aged F344 rats, the incidence of pheochromocytoma was greater in animals with more severe chronic progressive glomerulopathy (14). Nutritional factors have an important modulating effect on the spontaneous incidence of adrenal m edullary proliferative lesions in rats (12). Several sugars and sugar alcohols have produced adrenal m edullary tumors at high dosages, including xylitol, sorbitol, lacitol, and lactose at concentrations of 10 – 20% in the diet. Although the m echanism involved is not completely understood, a role for calcium has been postulated. High doses of slowly absorbed sugars and starches increase the absorption and urinar y excretion of calcium. In addition, vitamin D 3 induces proliferative lesions (hyperplastic nodules and pheochromocytomas) in the rat (21). Hypercalcemia is known to increase catecholamine synthesis in response to stress, and low-calcium diets will reduce the incidence of adrenal medullar y tumors in xylitol-treated rats. Other com pounds that m ight act via altered calcium homeostasis include the retinoids (which will produce hypercalcemia) and conditions such as progressive nephrocalcinosis in aging male rats treated with nonsteroidal anti-in ammator y agents. R EFERENCES 1. Capen CC, DeLellis RA, Yarrington JT (2001). Endocrin e system . In: Handboo k of Toxicologic Pathology, Hasek-Hock WM, Rousseaux MA, Wallig MA (eds). Academ ic Press, San Diego, in press. 2. Chen-Pan C, Pan IJ, Yam amoto Y, Sakogaw a T, Yamada J, Hayash i Y (1999). Prompt recovery of damaged adrenal medullae induced by salinomycin. Toxicol Pathol 27: 563 – 572. 3. Colby HD, Longhurst PA (1992). Toxicology of the adrenal gland. In: Endocrine Toxicology, Atterwill CK, Flack JD (eds). Cambridge University Press, Cambridge, pp 243 – 281.

47

4. Frith CH (1996). Lipogenic pigmentation, adrenal cortex, mouse. In: The Endocrin e System, ILSI Monograph s on Laboratory Animals, Jones TC, Mohr U, Capen CC (eds). Springer-Verlag, Heidelberg, pp 458 – 462. 5. Frith CH, Botts S, Jokinen MP, Eighm y JJ, Hailey JR, M organ SJ, Chandra M (2000). Non-proliferative lesions of the endocrine system in rats. In: Guides for Toxicologic Pathology. STP/ARP/AFIP, Washington, DC, pp 1 – 22. 6. Gliatto JM, Alroy J, Schelling SH, Engler SJ, Dayal Y (1995). A light m icroscopic al, ultrastructural and imm unohistochemical study of spindle-cell adrenocortical tumours of ferrets. J Comp Pathol 113: 175 – 183. 7. Goodman DG (1996). Subcapsular-cell hyperplasia, adrenal, mouse. In: The Endocrin e System, ILSI Monograph s on Laboratory Animals, Jones TC, Mohr U, Capen CC (eds). Springer-Verlag, Heidelberg, pp 464 – 467. 8. Hinson JP, Raven PW (2000). Adrenal Toxicology. In: Endocrine and Hormonal Toxicology, Harvey PW, Rush KC, Cockbur n A (eds). John Wiley & Sons, New York, pp 67 – 89. 9. Latendresse JR, Azhar S, Brooks CL, Capen CC (1993 ). Pathogenesis of cholesteryl lipidosis of adrenocortical and ovarian interstitial cells in F344 rats caused by tricresyl phosphate and butylated triphenyl phosphate . Toxicol Appl Pharmacol 122: 281 – 289. 10. Latendresse JR, Brooks CL, Capen CC (1994). Pathologic effects of butylated triphenyl phosphate- based hydraulic  uid and tricresyl phosphate on the adrenal gland, ovary, and testis in the Fischer344 rat. Toxicol Pathol 22: 341 – 352. 11. Latendresse JR, Brooks CL, Capen CC (1995). Toxic effects of butylated triphenyl phosphate- based hydraulic  uid and tricresyl phosphate in female F344 rats. Vet Pathol 32: 394 – 402. 12. Lynch BS, Tischler AS, Capen C, Munro IC, McGirr LM, McClain RM (1996). Low digestible carbohydra tes (polyols and lactose): SigniŽ cance of adrenal medullary proliferative lesions in the rat. Regul Toxicol Pharmaco l 23: 256 – 297. 13. M esiano S, Jaffe RB (1997). Developm ental and functional biology of the primate fetal adrenal cortex. Endocr Rev 18: 378 – 403. 14. Nyska A, Haseman JK, Hailey JR, Smetana S, Maronpot RR (1999). The association between severe nephropath y and pheochromocytoma in the male F344 rat— The National Toxicology Program experience. Toxicol Pathol 27: 456 – 462. 15. Nyska A, Maronpot RR (1999). Adrena l Gland. In: Pathology of the Mouse, Maronpot RR, Boorman GA, Gaul BW (eds). Cache River Press, Vienna, Illinois, pp 509 – 536. 16. Parker GA, Valerio MG (1996). Lipogenic pigmentation, adrenal cortex, rat. In: The Endocrine System, ILSI Monograph s on Laboratory Animals, Jones TC, M ohr U, Capen CC (eds). SpringerVerlag, Heidelberg, pp 462 – 464. 17. Ribelin W E (1984). The effects of drugs and chemicals upon the structure of the adrenal gland. Fundam Appl Toxicol 4: 105 – 119. 18. Sass B (1996). Am yloidosis, adrenal, mouse. In: The Endocrine System, ILSI M onograph s on Laboratory Animals, Jones TC, Mohr U, Capen CC (eds). Springer-Verlag, Heidelberg , pp 455 – 458. 19. Tischler AS (1996). Toxic respons e of the adrenal medulla. In: Comprehensive Toxicology, Sipes IG, McQueen CA, GandolŽ AJ (eds). Elsevier Science, Oxford, pp 651 – 669. 20. Tischler AS, DeLellis RA, Perlman RL, Allen JM , Costopoulo s D, Lee YC, Nunnemacher G, Wolfe HJ, Bloom SR (1985). Spontaneous proliferative lesions of the adrenal medulla in aging LongEvans rats: Com parison to PC12 cells, small granule-containing cells, and human adrena l medullary hyperplasia. Lab Invest 53: 486 – 498. 21. Tischler AS, Powers JF, Pignatello M, Tsokas P, Downing JC, McClain RM (1999). Vitamin D 3-induced proliferative lesions in the rat adrenal medulla. Toxicol Sci 51: 9 – 18. 22. Tischler AS, Powers JF, Shahsavari M, Ziar J, Tsokas P, Downing J, McClain RM (1997). Comparative studies of chromafŽ n cell proliferation in the adrenal medulla of rats and m ice. Fundam Appl Toxicol 35: 216 – 220. 23. Tischler AS, Ruzicka LA, Van Pelt CS, Sandusky GE (1990). Catecholamine-synthesizing enzymes and chromogranin proteins in

Downloaded from tpx.sagepub.com by guest on May 24, 2011

48

ROSOL ET AL

drug-induc ed proliferative lesions of the rat adrenal medulla. Lab Invest 63: 44 – 51. 24. Tischler AS, Ziar J, Downing JC, M cClain RM (1995). Sustained stimulation of rat adrenal chromafŽ n cell proliferation by reserpine . Toxicol Appl Pharmacol 135: 254 – 257. 25. Woodman DD (1997). The adrenal glands. In: Laboratory Animal Endocrinology, Woodman DD (ed). John Wiley & Sons, New York, pp 253 – 286. 26. Yarrington JT, Huffman KW, Gibson JP (1981). Adrenocortical degeneration in dogs, monkeys, and rats treated with alpha-(1,4-dioxido-3-m ethylquinoxa lin-2-yl)-N-methylnitrone. Toxicol Lett 8: 229 – 234.

T OXICOLOGIC P ATHOLOGY

27. Yarrington JT, Latendresse JR, Capen CC (1996). Toxic responses of the adrenal cortex. In: Comprehe nsive Toxicology, Sipes IG, M cQueen CA, GandolŽ AJ (eds). Elsevier Science, Oxford, pp 637 – 649. 28. Yarrington JT, Loudy DE, Sprinkle DJ, Gibson JP, Wright CL, Johnston JO (1985). Degenerat ion of the rat and canine adrenal cortex caused by alpha-(1,4- dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone (DMNM). Fundam Appl Toxicol 5: 370 – 381. 29. Yarrington JT, Reindel J (1996). Chemically-induced adrenocortical degenerative lesions. In: The Endocrine System, ILSI Monograph s on Laboratory Animals, Jones TC, Mohr U, Capen CC (eds). Springer-Verlag, Heidelberg, pp 467 – 476.

FUTURE STP SYMPOSIA 2001

June 25 – 28 Hyatt Orlando, FL Toxicologic Patholog y in the New M illenium

2002

June 17 – 20 Antlers Adams M ark Hotel Colorado Springs, CO Non-Rodent Species in Toxicologic Pathology

2003

June 16 – 19 W estin Savannah Harbor Resort Savannah, GA Toxicologic Patholog y and Carcinogenesis

2004

June 14 – 17 The Grand America Salt Lake City, UT Toxicologic Patholog y of Organs of Special Senses

Downloaded from tpx.sagepub.com by guest on May 24, 2011