Disruption of Glucocorticoid and Mineralocorticoid Receptor-Mediated Responses by Environmental Chemicals

ENDOCRINE DISRUPTORS: BASIC RESEARCH AND METHODS 335 doi:10.2533/chimia.2008.335 CHIMIA 2008, 62, No. 5

Chimia 62 (2008) 335–339 © Schweizerische Chemische Gesellschaft ISSN 0009–4293

Alex Odermatt* and Christel Gumy

Abstract: Glucocorticoids and mineralocorticoids are key endocrine hormones modulating essential physiological processes such as energy metabolism, cell growth and differentiation, maintenance of blood pressure and immune responses. Despite their importance and the fact that their impaired function has been associated with various diseases, there are only few studies on the potential disruption of glucocorticoid and mineralocorticoid action by xenobiotics. To facilitate the identification and characterization of such chemicals, we established cell-based assays to determine the impact of xenobiotics on different steps of corticosteroid hormone action. Screening of a small library of chemicals led to the identification of several compounds inhibiting the 11β -hydroxysteroid dehydrogenase (11β -HSD) prereceptor enzymes 11β -HSD1 and/or 11β -HSD2 and of chemicals blocking the function of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). These findings build a basis to extend the search for chemicals acting on additional targets of the corticosteroid hormone pathway and to apply in silico prediction tools in combination with biological testing to screen large numbers of chemicals. The identification of chemicals interfering with corticosteroid action and the elucidation of the underlying molecular mechanisms are relevant with respect to the potential contribution to common diseases such as metabolic syndrome, immune diseases, brain disorders and cancer. Keywords: Cortisol · Endocrine disruptor · Glucocorticoid receptor · Glucocorticoid · 11β -Hydroxysteroid dehydrogenase · Mineralocorticoid receptor · Xenobiotics

1. Physiological Role cell cycle control, and modulation of stress are produced in the adrenal cortex involv- of Glucocorticoids and and inflammatory responses (Fig. 1).[1] An ing several steroidogenic enzymes, and Mineralocorticoids impaired regulation of glucocorticoid ac- the synthesis of these hormones is tightly tion has been associated with various com- regulated by corticotrophin releasing fac- Corticosteroid hormones are divided into plications including metabolic and cardio- tor (CRF) and adrenocorticotrophic hor- glucocorticoids (cortisol in humans and vascular diseases, osteoporosis, cataracts, mone (ACTH) via the hypothalamus-pitu- corticosterone in rodents) and mineralocor- immune diseases, mood and cognitive dis- itary-adrenal (HPA) axis. Glucocorticoids ticoids (aldosterone). They are involved in orders and cancer. [2− 5] Disturbances of min- control their own synthesis by a negative the regulation of many physiological pro- eralocorticoid action have been linked to feedback response mediated by inhibition cesses, including energy metabolism, elec- the occurrence of hypertension and cardio- of CRF and ACTH. Upon release into the trolyte and blood pressure control, bone vascular diseases.[6,7] The incidence of these blood stream, corticosteroid hormones are metabolism, regulation of brain function, complex diseases increases with increasing mainly bound to carrier proteins (transcor- age, and in addition to genetic predispo- tin, albumin) and reach cells in peripheral sition, factors including life style and the tissues. exposure to xenobiotics are likely to play At the cellular level, cortisol or corti- a role in these pathological processes. In de- costerone exert their action through GR and veloped countries, the exposure to chemi- aldosterone acts by activating MR. Impor- cals from the environment may contribute tantly, the local activation of the receptors is to the high incidence of allergic diseases, controlled by two distinct 11β -HSD prere- cancer and metabolic disturbances.[8− 10] ceptor enzymes.[11] 11β -HSD1 is expressed ubiquitously and catalyzes predominantly * Correspondence: Dr. A. Odermatt the reduction of inactive 11-ketoglucocor- Molecular and Systems Toxicology 2. Major Targets of the ticoids (cortisone, 11-dehydrocortisone) Department of Pharmaceutical Sciences University of Basel Corticosteroid Hormone Pathway into active 11β -hydroxyglucocorticoids Klingelbergstrasse 50 (cortisol, corticosterone).[12] This enzyme CH-4056 Basel Exogenous chemicals can cause distur- has a crucial role in potentiating local GR Tel.: +41 61 267 1530 Fax: +41 61 267 1515 bances of corticosteroid hormone action activation in metabolic processes and in the E-mail: [email protected] at several steps (Fig. 2). Corticosteroids immune system. ENDOCRINE DISRUPTORS: BASIC RESEARCH AND METHODS 336 CHIMIA 2008, 62, No. 5

fact that they belong to different classes of proteins and share very low sequence similarity. This suggests that the binding pockets of these proteins share structural similarity and that the chemicals mimicking corticosteroid molecules might interact with more than one of these proteins. Thus, to assess the potential disruption of corticosteroid hormone action by exogenous chemicals, suitable bioassays are required to measure the activities of the different proteins, with roles in regulation, biosynthesis, transport, intracellular metabolism, receptor and degradation.

3. Bioassays

Compared to the extensively studied field of estrogen- and androgen-like ac- tions by environmentally relevant chemicals, there are only few studies focusing on corticosteroid hormones.[16] As a start- ing point to identify chemicals that act on Fig. 1. Overview of physiological functions that are regulated by corticosteroid hormones different steps of glucocorticoid and mineralocorticoid regulation, we established assays to measure the function of human corticosteroid hormone receptors (GR and MR) and glucocorticoid metabolizing enzymes (11β -HSD1 and 11β -HSD2). To distinguish between different steps of receptor activation, the HEK-293 cell line was selected that is devoid of endogenous expression of corticosteroid receptors and hormone metabolizing enzymes.[17] Other cells that are suitable alternatives include COS-1 and CV-1 cells,[18] but they are not of human origin. Receptor activity was measured in intact cells transiently expressing recombinant receptor or a green-fluorescence (GFP)- chimeric receptor. This allowed the assess- ment of the effect of a given chemical on ligand binding to the receptor, subsequent translocation of the receptor into the nucleus and receptor-mediated transcriptional activation of a reporter gene. It is important to distinguish the different steps of receptor activation to understand the inhibitory Fig. 2. Schematic overview of targets of corticosteroid hormone action mechanism of a chemical. Spironolactone, as an example, efficiently binds to MR and induces translocation of the receptor into the nucleus. It acts as a partial agonist and The second enzyme, 11β -HSD2, is tects the fetus from high maternal gluco- therefore antagonizes the potent agonist expressed in cortical collecting ducts corticoid concentrations.[15] effect of aldosterone. The bile acid cheno- and distal tubules in the kidney and The tissue-dependent responses upon deoxycholic acid inhibits 11β -HSD2 and in distal colon, where it protects MR activation of GR and MR strongly depend leads to glucocorticoid-induced activation from glucocorticoids by converting on the presence of coactivator and corepres- of MR. Although it also induces nuclear 11β -hydroxyglucocorticoids into 11-keto- sor proteins that interact with the receptor translocation of MR in the absence of glucocorticoids.[13] The MR has similar complex, as well as on post-translational 11β -HSD2, chenodeoxycholic acid neither affinities to bind aldosterone and cortisol modifications of both receptor and associ- activates nor antagonizes MR activation by and circulating concentrations of the latter ated proteins. Finally, enzymes responsible cortisol or aldosterone.[19,20] are 100− 1000 times higher, thus, the close for the degradation and excretion of the ste- To assess the effect of chemicals on proximity of 11β -HSD2 to the receptor al- roid hormones are important to terminate 11β -HSD prereceptor enzyme function, lows aldosterone to bind.[14] 11β -HSD2 is hormone action. the conversion of cortisone to cortisol or the also expressed in the syncytiotrophoblast The proteins described above all recog- reverse reaction was measured in lysates or layer of the human placenta, where it pro- nize corticosteroid molecules despite the intact HEK-293 cells stably expressing C- ENDOCRINE DISRUPTORS: BASIC RESEARCH AND METHODS 337 CHIMIA 2008, 62, No. 5 terminally FLAG-tagged human 11β -HSD1 identified, compounds are responsible for with 11β -HSD2 function by modification or 11β -HSD2.[21] The impact of 11β -HSD inhibition of 11β -HSD2. Additional com- of cysteine residues. Dithiothreitol, but enzyme inhibition on receptor function pounds of the triterpenoid and flavonoid not glutathione, protected from organotin- was determined in cells cotransfected with class of chemicals that inhibited 11β -HSD2 dependent inhibition. Enhanced glucocor- 11β -HSD1 and GR or 11β -HSD2 and MR, include abietic acid, gossypol, magnolol ticoid concentrations, due to disruption of respectively. As controls for 11β -HSD1 and and tea polyphenols. However, their rela- 11β -HSD2 function, may contribute to the 11β -HSD2 the well known inhibitor glycyr- tively weak activities observed in intact observed organotin-dependent toxicity in rhetinic acid was used, and for MR and GR, cell assays suggest that these compounds some glucocorticoid-sensitive tissues such spironolactone and RU486 were applied to are unlikely to cause physiological conse- as thymus and placenta. Reduced birth optimize assay conditions. quences by inhibiting 11β -HSD2.[33] weight and thymus involution, observed The strategy of expressing recombinant We then tested about 100 environmen- upon exposure to organotins, can also be corticosteroid receptors in the presence of tally relevant chemicals that were selected caused by excessive glucocorticoid levels. modulatory proteins to identify chemicals based on evidence in the literature for po- disrupting corticosteroid action by indirect tential interference with corticosteroid ac- mechanisms will now be extended. The tion, for their effect on 11β -HSD2 activity. 5. Chemicals Disrupting MR impact on receptor function of chemicals In addition to the compounds mentioned Activation interfering with signaling pathways is cur- above, abietic acid, fusidic acid, zearale- rently under investigation. For the efficient none, 4-tert-octylphenol, 4-nonylphenol, The same set of about 100 chemicals analysis of chemicals interfering with cor- bisphenol A, endosulfan and several di- was screened using the MR nuclear trans- ticosteroid hormone action, several cell thiocarbamates and organotins inhibited location assay to test them for agonist or an- models stably expressing different combi- 11β -HSD2.[21] Among these, abietic acid, tagonist properties. Aldosterone-mediated nations of receptor and modulatory protein bisphenol A, dithiocarbamates and organo- nuclear translocation of MR was inhibited will be constructed. tins inhibited 11β -HSD2 at subcytotoxic by various chemicals with different prop- concentrations. Dithiocarbamates and or- erties, including bisphenol A, endosulfan, ganotins were studied in more detail. 4-nonylphenol, vinclozolin, zearalenone, 4. Compounds Inhibiting 11β -HSD2 Dithiocarbamate chemicals are widely and some phthalate derivatives. All of these used in agriculture as pesticides or fungi- chemicals inhibited nuclear translocation Liquorice is a well-known endocrine cides and as vulcanization accelerators in of MR at high concentrations of 20− 50 µ M disruptor inducing corticosteroid hormone latex production (mainly in gloves). Dithio- that are unlikely to be physiologically rel- responses.[22] Glycyrrhetinic acid, which is carbamates are considered to be responsi- evant. The screening of this small number contained in liquorice, has been identified ble for the allergic reactions against rub- of chemicals showed that chemicals inter- as a potent inhibitor of 11β -HSD enzymes. ber products.[34] Interestingly, disulfiram, fering with MR activation exist, although By inhibiting 11β -HSD2, glycyrrhetinic known asAntabus to treat alcoholic patients, none of the identified compounds was of acid prevents inactivation of cortisol in was also active. Among the dithiocarbam- high potency. It cannot be excluded at this cortical collecting ducts and distal tubules ates analyzed, disulfiram and thiram were point that some of the identified chemicals in the kidney, thereby leading to cortisol- most potent with an IC50 value of 130 nM, have more pronounced effects under certain induced MR activation, sodium retention followed by maneb, diethyldithiocarbamate conditions such as oxidative stress or upon and hypertension.[14,23− 26] 11β -HSD2 also and zineb. [35] The inhibitory potential of preincubation for a prolonged time. Clearly, has a key function in the regulation of fetal disulfiram and thiram to inhibit 11β -HSD2 a more extensive screening will be neces- development[15] and protects the fetus from was comparable with that for aldehyde de- sary for the identification of chemicals with the high maternal cortisol concentrations. hydrogenase. These chemicals irreversibly physiologically relevant effects. The intrauterine exposure to elevated levels inhibited 11β -HSD2, probably by covalent The contribution of dithiocarbamate- of glucocorticoids has been associated with carbamoylation of catalytically important dependent 11β -HSD2 inhibition to cor- reduced birth weight and glucose intoler- cysteine residues. Glutathione protected tisol-induced activation of corticosteroid ance and cardiovascular complications later 11β -HSD2 from inhibition by dithiocar- receptors is unclear, since we found that in life,[27,28] and evidence has been provided bamates, suggesting that these chemicals high concentrations of these chemicals also that this programming is altered through are most critical in situations of oxidative could block MR and GR. This is in line with several generations.[29] This indicates a role stress, when intracellular glutathione con- a recent study by Garbrecht et al. who de- for epigenetic factors in the transgenera- centrations are low. The exposure to the di- scribed thiram-dependent inhibition of GR tional information transfer of these altera- thiocarbamates maneb and zineb has been activation.[37] An inhibition of 11β -HSD2 tions. The inhibition of 11β -HSD2 during associated with acute renal failure and and/or MR and GR is only likely to occur pregnancy by glycyrrhetinic acid or its ana- nephrotic syndrome in agricultural work- under conditions of glutathione depletion, logue carbenoxolone leads to elevated glu- ers, as well as kidney damage and reduced and further studies are required to see which cocorticoid levels and causes reduced birth body weights in the offspring from exposed protein is inhibited at the lowest concentra- weight with a higher risk for cardiovascu- pregnant rats. The inhibition of 11β -HSD2 tions. lar disease later in life.[25] The inhibition of may contribute to some of the observed 11β -HSD2 by chemicals from the environ- toxic effects of these chemicals in kidney ment in a critical window during pregnancy and on blood pressure, as well as in pla- 6. Compounds Inhibiting 11β -HSD1 is thus expected to cause detrimental effects centa and on fetal development. and should be considered as a risk factor for We identified several organotins includ- Inhibition of 11β -HSD1-dependent metabolic and cardiovascular diseases. ing trialkyltins and dialkyltins that potently glucocorticoid activation is currently con- Grape fruit juice has also been associ- inhibited 11β -HSD2 but not 11β -HSD1, sidered as a promising strategy to treat pa- ated with inhibition of 11β -HSD2,[30,31] 17β -HSD1 or 17β -HSD2.[36] They revers- tients with metabolic syndrome[5] and many and naringenin was suggested as the active ibly inhibited the enzyme with comparable medium and large pharmaceutical compa- compound. However, naringenin is a weak potencies in assays with cell lysates or in- nies have inhibitor development programs. [21,32] 11β -HSD2 inhibitor (IC50 > 300 µ M), tact cells. Analysis of the inhibitory mech- While preventing excess production of ac- and it is more likely that other, as yet un- anism suggested that organotins interfere tive glucocorticoids is important to avoid ENDOCRINE DISRUPTORS: BASIC RESEARCH AND METHODS 338 CHIMIA 2008, 62, No. 5 adverse metabolic effects and inhibition of metabolizes 7-ketodehydroepiandrosterone activities of metabolizing enzymes and re- 11β -HSD1 is beneficial in such situations, and 7-ketopregnenolone, suggesting a role ceptor function. The development of suit- glucocorticoid deficiency has been associ- not only in the detoxification of oxidized able in silico prediction tools and systemic ated with impaired immune responses in cholesterol from food but also of oxidized approaches, including analyses of effects inflammatory reactions[38] and a reduced steroid hormone metabolites.[49] of xenobiotics on the transcriptome and 11β -HSD1 expression has been found in proteome, is necessary for the identifica- some forms of cancer. [39,40] Thus, depend- tion of chemicals disrupting corticosteroid ing on the tissue and on the metabolic state, 7. Interference of Chemicals with hormone action and to understand their inhibition of 11β -HSD1 may have different GR Function mechanisms of action. consequences. Several compounds inhibiting 11β - The same set of about 100 chemicals Acknowledgements HSD1 were identified upon screening was screened using our nuclear GR trans- This work was supported by grants from our small library. Zearalenone, 4-tert- location assay. Bisphenol A, zearalenone the Swiss National Science Foundation No octylphenol, 4-nonylphenol, methyljas- and some phthalate derivatives inhibited 310000-112279 and NRP50 ‘Endocrine monate, dibenzoylmethane and 2,2’-dihy- cortisol-induced nuclear translocation of Disruptors’ No 4050-066575. A.O. is a Novartis Research Foundation Professor. droxybiphenyl were weak inhibitors, un- GR. These chemicals also inhibited nuclear [21] likely to be of physiological relevance. MR translocation. BisphenolA and zearale- Received: March 22, 2008 Abietic acid and flavanone as well as some none inhibited the function of all four pro- monohydroxylated flavanone derivatives teins tested, suggesting either an unspecific were more potent. While abietic acid dis- mechanism or that these chemicals mimic [1] R. M. Sapolsky, L. M. Romero, A. U. played a similar activity as the one observed the steroid hormone cortisol and are there- Munck, Endocr. Rev. 2000, 21, 55. [2] M. C. Carnahan, D. A. Goldstein, Curr. with glycyrrhetinic acid and carbenox- fore recognized by all four proteins. In ad- Opin. Ophthalmol. 2000, 11, 478. olone and inhibited both 11β -HSD1 and dition, dibenzoylmethane and abietic acid [3] J. K. Belanoff, K. Gross, A. Yager, A. F. 11β -HSD2, flavanone and its derivatives at concentrations of 20− 50 µ M were shown Schatzberg, J. Psychiatr. Res. 2001, 35, selectively inhibited 11β -HSD1 by com- to inhibit GR translocation. Due to the high 127. peting with substrate binding. Analysis of concentrations required for inhibition, a di- [4] J. R. Seckl, Eur. J. Endocrinol. 2004, 151 the structural requirements for the inhibi- rect effect on GR was considered not physi- Suppl 3 , U49. tory effect of flavanones revealed that hy- ologically relevant. In these experiments, [5] A. G. Atanasov, A. Odermatt, Endocr. droxylation at position 6 leads to reduced effects on transcriptional expression or on Metab. Immune Disord. Drug Targets potency. 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