Toxicology VOL. VOL. 26 26 about the book… TARGET ORGAN TOXICOLOGY T A R G E T O R G A N T O X I C O L O G Y S E R I E S Despite being regarded as the most common toxicological target in the endocrine system, SERIES the adrenal gland has often been neglected in regulatory testing. Adrenal Toxicology addresses the increased interest in adrenocortical toxicology and the need for a resource Series Editors that makes techniques available to examine adrenal endocrine disruption. A. Wallace Hayes • John A. Thomas • Donald E. Gardner Examining current techniques and the latest advancements, Adrenal Toxicology reviews A the endocrinology, pharmacology, pathology and toxicology of the adrenal gland. This text provides information on the range of drugs and chemicals that affect adrenocortical function and suggests standardized approaches for in vivo and in vitro assessment. This d volume also presents recent developments in the molecular mechanisms of toxicity to the r adrenal cortex and medulla, and considers environmental adrenal endocrine disruption in sentinel species. e Adrenal Toxicology: n • reflects the major developments made over the past decade • focuses on the latest research techniques, including their uses and limitations A • provides an integrated strategy for adrenal toxicology evaluation l • identifies knowledge and data gaps, providing impetus for regulatory consideration A about the editors... T drenAl PHILIP W. HARVEY received his honors degree and doctorate (endocrine, reproductive/ developmental, and neurobehavioral toxicology) from the University of Keele, UK, and has o worked in general industrial/pharmaceutical toxicology for over 20 years. He has particular x interests in endocrine toxicology (the adrenal, prolactin and hormonal carcinogenesis, T estrogenic chemicals and human health) and devised and edited the books The Adrenal in i oxicology c Toxicology: Target Organ and Modulator of Toxicity (1996) and Endocrine and Hormonal Toxicology (1999). He has served on various journal editorial boards and is currently the o Editor-in-Chief of Journal of Applied Toxicology. l DAVID J. EVERETT received his honors degree in biological sciences from the University of Sussex, UK, and has been actively involved in toxicological safety assessment of novel o pharmaceuticals, agrochemicals and industrial chemicals for more than 30 years. His g involvement in carcinogenicity has led to a particular interest for him in hormonal carcinogenesis. He is a Fellow of the Institute of Biology. y CHRISTOPHER J. SPRINGALL is a toxicological pathologist who received his original medical degree from Liverpool University, UK, and has been actively involved in toxicology and drug development for over 25 years. He has worked with a very wide range of candidate drugs, including a substantial number which have an impact on endocrine systems. Edited by Printed in the United States of America Harvey Philip W. Harvey Everett David J. Everett Springall 61291 Christopher J. Springall A drenAl T oxicology TARGET ORGAN TOXICOLOGY SERIES SeriesEditors A.WallaceHayes,JohnA.Thomas,andDonaldE.Gardner AdrenalToxicology.PhilipW.Harvey,DavidJ.Everett,andChristopherJ. Springall,editors,336pp.,2008 CardiovascularToxicology,FourthEdition.DanielAcosta,Jr.,editor,712pp., 2008 ToxicologyoftheGastrointestinalTract.ShayneC.Gad,editor,384pp.,2007 ImmunotoxicologyandImmunopharmacology,ThirdEdition.RobertLuebke, RobertHouse,andIanKimber,editors,676pp.,2007 ToxicologyoftheLung,FourthEdition.DonaldE.Gardner,editor,696pp.,2006 ToxicologyofthePancreas.ParvizM.Pour,editor,720pp.,2005 ToxicologyoftheKidney,ThirdEdition.JoanB.TarloffandLawrenceH.Lash, editors,1200pp.,2004 OvarianToxicology.PatriciaB.Hoyer,editor,248pp.,2004 CardiovascularToxicology,ThirdEdition.DanielAcosta,Jr.,editor,616pp., 2001 NutritionalToxicology,SecondEdition.FrankN.KotsonisandMaureenA. Mackey,editors,480pp.,2001 ToxicologyofSkin.HowardI.Maibach,editor,558pp.,2000 Neurotoxicology,SecondEdition.HughA.TilsonandG.JeanHarry,editors, 386pp.,1999 Toxicant–ReceptorInteractions:ModulationofSignalTransductionsandGene Expression.MichaelS.DenisonandWilliamG.Helferich,editors,256pp.,1998 ToxicologyoftheLiver,SecondEdition.GabrielL.PlaaandWilliamR.Hewitt, editors,444pp.,1997 FreeRadicalToxicology.KendallB.Wallace,editor,454pp.,1997 EndocrineToxicology,SecondEdition.RaphaelJ.Witorsch,editor,336pp., 1995 Carcinogenesis.MichaelP.WaalkesandJerroldM.Ward,editors,496pp., 1994 DevelopmentalToxicology,SecondEdition.CaroleA.KimmelandJudy Buelke-Sam,editors,496pp.,1994 NutritionalToxicology.FrankN.Kotsonis,MaureenA.Mackey,andJerryJ. Hjelle,editors,336pp.,1994 OphthalmicToxicology.GeorgeC.Y.Chiou,editor,352pp.,1992 ToxicologyoftheBloodandBoneMarrow.RichardD.Irons,editor,192pp., 1985 ToxicologyoftheEye,Ear,andOtherSpecialSenses.A.WallaceHayes,editor, 264pp.,1985 CutaneousToxicity.VictorA.DrillandPaulLazar,editors,288pp.,1984 A drenAl T oxicology Edited by Philip W. Harvey Covance Laboratories UK Ltd. North Yorkshire, UK David J. Everett Covance Laboratories UK Ltd. North Yorkshire, UK Christopher J. Springall Covance Laboratories UK Ltd. North Yorkshire, UK InformaHealthcareUSA,Inc. 52VanderbiltAvenue NewYork,NY10017 (cid:1)C 2009byInformaHealthcareUSA,Inc. InformaHealthcareisanInformabusiness NoclaimtooriginalU.S.Governmentworks PrintedintheUnitedStatesofAmericaonacid-freepaper 10987654321 InternationalStandardBookNumber-10:1-4200-6129-1(Hardcover) InternationalStandardBookNumber-13:978-1-4200-6129-1(Hardcover) This book contains information obtained from authentic and highly regarded sources. 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AdrenalGlands–drugeffects. 2.AdrenalGlands–physiology. 3.AdrenalCortexDiseases–chemicallyinduced. 4.AdrenalCortex Diseases–physiopathology. 5.Toxicology–methods.WK702A24272008] RC659.A3752008 616.4’5–dc22 2008020500 ForCorporateSalesandReprintPermissionscall212-520-2700orwriteto:SalesDepartment, 52VanderbiltAvenue,7thfloor,NewYork,NY10017. VisittheInformaWebsiteat www.informa.com andtheInformaHealthcareWebsiteat www.informahealthcare.com For my family in England and Slovakia, especially my wife Daniela and our childrenJessicaRuth,RebeccaEve,andWilliamJozef. P.W.H. Formyfather,John. D.J.E. For colleagues at Covance whose support has allowed us the time to undertake thisproject. C.J.S. Preface Despite the adrenal gland being the most common target within the endocrine system (Ribelin, 1984), adrenal dysfunction is poorly recognized in toxicology. Theadrenalisadefinedvitalorganwithaprimaryroleintheadaptationtostress- ful 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 regula- torystudiesoftendisregardsadrenocorticalhistologicalfindingsassecondaryto stress, which can be inappropriate without evidence that the adrenal cortex is functionally competent. For example, adrenal hypertrophy may certainly be due tostress-inducedoversecretionofadrenocorticotropichormone(ACTH),butthis conditionmayalsoarisefrommoreseriousadrenocorticalsteroidogenicenzyme inhibition,theconsequentlossofglucocorticoid(cortisolorcorticosterone)secre- tion,andreducedorabolishedfeedbackcontrolofpituitaryACTHsecretion.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 steroidogene- siscanbeaseriousconditionleadingtoAddisoniancrisis(adrenalinsufficiency characterizedbylethargy,hemodynamicinstability,andcardiovascularcollapse) and death. Indeed, there are many examples of drugs and chemicals known to inhibit critical adrenocortical enzymes, potentially producing adrenal incompe- tence,insufficiency,orsuppression,andseveralexampleshavebeendiscoveredin patientsfollowingunexpectedside-effectsandfatalitiesapparentlynotadequately detected,orindeedignoredasinconsequential,inpreclinicaltoxicology. 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, inflam- mation,immunefunction,andvariousreproductiveanddevelopmentalprocesses 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 criticalomissioninaregulatorytoxicologycontext (HarveyandJohnson,2002; Harvey and Everett, 2003; Harvey and Everett, 2006; Hinson and Raven, 2006; Harvey, Everett, and Springall, 2007). The United States Environmental Pro- tection Agency (USEPA), Endocrine Disrupter Screening and Testing Advisory v vi Preface 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 H295Rcelllinehasbeensuggestedasausefulsystemtoaddressbothissues(e.g., HarveyandEverett,2003;Harvey,Everett,andSpringall,2007).Otherregulatory bodieshavesincepartiallyrectifiedthissituationandrecognizedtheimportance oftheadrenalintoxicology,oratleasttheutilityofadrenocorticalcellsasamodel forsteroidogenesisasawhole,andtheOECDhasrecentlyundertakenaprogram tovalidatetheH295Rcelllineasauniversalmodeltoevaluatesteroidogenictox- icity(e.g.,Heckeretal.,2007).Oskarssonetal.(2006)reportthatsteroidogenic geneexpressionfollowingchemicalchallengeiscomparablebetweentheH295R celllineandthenormalhumanadrenaldemonstratingthevalidityofthiscellline. 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 challengeaugmentationcanbeachievedbydrugs,suchasforskolin. The number of literature studies on adrenocortical toxicity has risen markedlyoverthepastdecade,whichindicatesthegrowingscientificandregula- toryinterestintheadrenalasatargetfor“endocrinedisruption,”withthemajority of studies using in vitro techniques. Numerous laboratories are now using the H295Rcelllinetoinvestigatethediverseeffectsofchemicalsonmolecularmech- anisms of adrenocortical toxicity, and/or the general process of steroidogenesis, using steroid production, gene regulation, and enzyme expression as endpoints seeSandersonetal.,2001;Muller-Vieiraetal.,2005;Graciaetal.,2006;Hecker etal.,2006;Oskarssonetal.,2006;Furutaetal.,2008andStiglianoetal.,2008 for recent examples of the range of endpoints that can be assessed in this cell line,andchapters7and8forthoroughreviews).Whilethisisanimportantstep 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, andthereforethereisalsoaneedtovalidateinvivomodels.Astrategyforevalu- ationofdrugandchemicaleffectsonadrenocorticalfunctionhasbeenproposed (Harvey,Everett,andSpringall,2007,andalsoseechap.1forfurtherdetails)and involvesashort-terminvivoadrenalchallengetestinrodents(orotherlaboratory speciesifindicated—seeColagiovannietal.,2006,andinthisvolume,forinfor- mation 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. Theprimarypurposeofthistextistoreviewthescopeof,anddevelopments 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 Preface vii OrganandModulatorofToxicity(Harvey,1996a).Thepresenttexthasminimal overlap with the former, but reviews the major developments in the field over the last decade, and identifies research requirements including the validation of standardizedmodelsandmethods.Thefocusofthisnewtextisontheadrenalas a target organ, both in the main mammalian species used in pharmaceutical and chemical regulatory toxicology and environmental sentinel species, and also on theadvancesinidentifyingmolecularmechanismsofaction.Mosttoxicological researchhasfocusedonthecortexratherthanthemedulla,whichreflectsitswider and more complex role in physiological processes, and this is also by necessity reflectedinthistext,althoughpathologyofthemedullaisexaminedintherodent andhuman. 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 endocrinologyandpharmacologyoftheadrenalinhealthanddisease.Anexem- plary chapter on human adrenal dysfunction (see chap. 2) reinforces the critical importanceofthecorrectfunctionofthisgland,bothmedullaandcortex,andfrom this,thepotentialconsequencesofdrugorchemical-induceddysfunctionmaybe inferred.Forexample,manyadrenaldiseasesandsyndromesaregenerallydueto 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 adrenocorticaldisorderscanbepharmacologicallymanipulated(HakkiandBern- hardt, 2006) raises the real possibility that these adrenal conditions could occur asaconsequenceofoff-targettoxicityfromdrugsandchemicals,bothinnormal subjects,butespeciallyinpatientspredisposedtodevelopthenaturaletiological factorsofthedisease,whereadditionalinsultmayaccelerateorprecipitateonset. Inaddition,thissectionalsoprovidesahighlyauthoritativereview(seechap.3) of the endocrinology, pharmacology and pathophysiology of the hypothalamo- pituitary-adrenal(HPA)axis,includingnewemergingknowledgeofdevelopmen- taleffectsofHPAhormones.Theearlylifeinfluenceofglucocorticoidhormones isanimportantpharmacologicalfieldwithimmensepotentialapplication;adrenal glucocorticoidanalogueshavewelldocumentedusestoacceleratematurationof premature/fetal lung, but have also long been known to have adverse effects on generalgrowthanddevelopmentintermsofteratogenicity(Hawkins,1983).New evidencesuggeststheymayalsobeassociatedwithadultsusceptibilitytomajor diseasessuchashypertension,type2diabetes,coronaryheartdisease,hyperlipi- demia and nervous system disturbance following early life exposure. The phar- macologyofglucocorticoidsandeffectsonimmuneandinflammatoryresponses isintroduced(propertieswhichhavebeenexploitedinoneofthemostimportant 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 classicalpharmacologyofadrenalglucocorticoidsandtheirsyntheticanalogues).