Molecular and Cellular Endocrinology 212 (2003) 4149

A rapid screening assay for inhibitors of 11 -hydroxysteroid dehydrogenases (11 -HSD): avanone selectively inhibits 11 -HSD1 reductase activity
Roberto A.S. Schweizer1 , Atanas G. Atanasov1 , Brigitte M. Frey, Alex Odermatt,2
Department of Clinical Research, Division of Nephrology and Hypertension, University of Berne, Freiburgstrasse 15, Berne 3010, Switzerland Received 22 January 2003; accepted 17 September 2003

Abstract A rapid screening assay for chemicals inhibiting 11 -hydroxysteroid dehydrogenase (11 -HSD) type 1 or type 2 using lysates from stably transfected cells was developed. Here, we tested a series of environmental chemicals for anti-11 -HSD activities. Inhibition of 11 -HSD2, which may cause cortisol-dependent activation of the mineralocorticoid receptor with sodium retention and hypertension, was observed for several compounds, with diethylcarbamate being the most potent inhibitor (IC50 6.3 M). Abietic acid inhibited both 11 -HSD1 (IC50 27 M for reduction and 2.8 M for oxidation) and 11 -HSD2 (IC50 12 M). Our results demonstrate for the rst time that avanone selectively inhibits 11 -HSD1 reductase activity: this enzyme being considered as essential for the local activation of glucocorticoids and representing a potential target for the therapeutic treatment of diabetes type 2. Flavanone and 2 -hydroxyavanone efciently inhibited reductive (IC50 18 and 10 M) but not oxidative activity. We observed a reduced inhibitory effect of hydroxylated avanone derivatives and of avones containing a double-bond between atom C2 and C3. Flavanone was specic for 11 -HSD1 and did not inhibit 11 -HSD2. Our results reveal that a variety of environmental compounds exert distinct inhibitory effects on 11 -HSD1 and 11 -HSD2, opening the possibility for selectively modulating local cortisone/cortisol availability in vivo. 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: 11 -Hydroxysteroid dehydrogenase; Endocrine disruptor; Environmental chemical; Glucocorticoid; Flavonoid; Inhibitor

1. Introduction Environmental chemicals such as herbicides, pesticides, plasticizers and PCBs, as well as food ingredients such as phytoestrogens that mimic endogenous hormone action, disturb the endocrine regulation of various biological processes. There is a fast growing literature on such compounds with effects on mechanisms regulated by sex steroid

Abbreviations: DMEM, Dulbecos modied Eagles medium; GR, glucocorticoid receptor; HEK, human embryonic kidney; 3 -HSD, 3 hydroxysteroid dehydrogenase; 11 -HSD, 11 -hydroxysteroid dehydrogenase; 17 -HSD, 17 -hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCB, polychlorated biphenyl; PVC, polyvinyl chloride; TLC, thin layer chromatography Corresponding author. Tel.: +41-31-632-9438; fax: +41-31-632-9444. E-mail address: alex.odermatt@dkf2.unibe.ch (A. Odermatt). 1 R.A.S.S. and A.G.A. contributed equally to the present study. 2 A.O. is a Clotta Research Fellow.

hormone receptors (reviewed in Hoyer, 2001; Lathers, 2002). Recent evidence indicated that disturbances of estrogen receptor-mediated responses by some environmental chemicals can be due to the interference with the control of intracellular hormone concentrations by inhibition of 17 -hydroxysteroid dehydrogenases (17 -HSD) (Krazeisen et al., 2001; Le Bail et al., 2001). In contrast to disturbed sex steroid-mediated effects, the interference of environmental chemicals and food ingredients with glucocorticoid-mediated responses has not been extensively investigated until now. The inappropriate regulation of glucocorticoid concentrations induces an array of glucocorticoid receptor (GR)-mediated metabolic effects including osteoporosis, weight gain, diabetes, and cataracts, as well as mineralocorticoid receptor (MR)-mediated effects including renal salt retention with hypertension, hypokalemia and heart failure (Barnes, 1998; Stewart and Krozowski, 1999; White et al., 1997). All these disease states show an age-dependent increasing frequency, and the accumulation of inappropriate

0303-7207/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.09.027

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Fig. 1. Reactions catalyzed by 11 -HSD enzymes and cofactors used.

GR and MR effects, due to receptor response modulation by endocrine disruptor compounds during life, might contribute to these pathological processes. 11 -HSD1 and 11 -HSD2 constitute pre-receptor mechanisms by controlling the ratio of the local concentration of biologically active 11 -hydroxyglucocorticoids, e.g. cortisol, to their inactive 11-keto forms, e.g. cortisone (Fig. 1) (Stewart and Krozowski, 1999; White et al., 1997). 11 -HSD1 catalyzes both oxidation of cortisol and reduction of cortisone in an intact cell system, however, it acts mainly as a reductase in vivo. 11 -HSD1 is essential for the local activation of GR. Recently, it was demonstrated that transgenic mice overexpressing 11 -HSD1 developed central obesity and metabolic syndrome (Masuzaki et al., 2001). Thus, 11 -HSD1 inhibitors may have benecial effects in the treatment of individuals with visceral obesity. Indeed, treatment of hyperglycaemic mice with a selective 11 -HSD1 inhibitor signicantly reduced blood glucose levels, suggesting the application of 11 -HSD1 inhibitor for the treatment of individuals with diabetes type 2 (Alberts et al., 2002; Barf et al., 2002). In contrast, 11 -HSD2 exclusively catalyzes the oxidation of cortisol, thereby preventing the activation of the MR by cortisol and rendering specicity of the MR for aldosterone (Stewart and Krozowski, 1999; White et al., 1997). Impaired 11 -HSD2 activity due to genetic mutations has a negative impact on human health by causing sodium retention that eventually results in hypertension (New and Wilson, 1999). A similar form of hypertension was observed after the prolonged and excessive intake of licorice, which contains the 11 -HSD2 inhibitor glycyrrhetinic acid (Ferrari et al., 2001; Gomez-Sanchez and Gomez-Sanchez, 1992; Lindsay et al., 1996). In the present study, we established a rapid screening assay to identify inhibitors of 11 -HSD enzyme activities. We screened a series of environmental chemicals and phyto-compounds for both inhibitory effect on oxidative and reductive activity of 11 -HSD1 as well as on oxidative activity of 11 -HSD2.

with 1 mg of lysate of HEK-293 cells expressing 11 -HSD2 in the presence of 1 mM NAD+ . Incubation was for 16 h at 37 C. The steroids were extracted with ethylacetate, separated by thin layer chromatography (TLC) (SIL G-25 UV254, Macherey-Nagel, Oensingen, Switzerland) using a solvent system of 9:1 (v/v) chloroform:methanol, and the band corresponding to cortisone was excised. The product was run for a second chromatographic purication step on the same TLC system. A total of 250 Ci of [1,2,6,7-3 H]-cortisone was recovered. G-418 sulfate was from Promega, Wallisellen, Switzerland. All other chemicals were from Fluka AG, Buchs, Switzerland and were of the highest grade available. 2.2. Construction of stably transfected cells Untransfected HEK-293 cells do not express 11 -HSD1 activity. 11 -HSD2 mRNA was detectable by RT-PCR, but activity was not detectable upon incubation of radiolabeled cortisol with cell lysate for 8 h. HEK-293 cells were transfected with the plasmid for expression of carboxy-terminally FLAG-epitope tagged 11 -HSD1 or 11 -HSD2, respectively, that were described previously (Odermatt et al., 1999). Transfected cells were selected by cultivation in presence of 800 g/ml of G-418. Non-resistant cells were removed by replacing the cell culture medium every third day for 3 weeks. From these cells, eight clones each were then selected and tested for protein expression, by immunouorescence analysis using mouse monoclonal anti-FLAG antibody M2 (Sigma) and goat anti-mouse antibody ALEXA 488, and 11 -HSD activity. All of the eight clones of either 11 -HSD1 or 11 -HSD2 transfected cells showed similar expression and activity of the corresponding FLAG-epitope tagged 11 -HSD construct. 2.3. 11-HSD activity assay HEK-293 cells stably transfected with either 11 -HSD1 or 11 -HSD2 were grown in 10 cm dishes to 90% conuence. Cells were rinsed once with phosphate-buffered saline and resuspended in 2 ml of ice-cold buffer TS2 containing 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2 , 250 mM sucrose, 20 mM TrisHCl, pH 7.4. For determination of oxidative activity of 11 -HSD enzymes, cells were lyzed by sonication and the cell lysate was diluted 1:12 in buffer TS2 (at 4 C). Reactions were carried out in 96-well optical PCR reaction plates (Applied Biosystems, Foster City, CA) and tubes were capped during the reaction to avoid evaporation. Reactions were started by simultaneously adding 10 l of cell lysate and 10 l of TS2 buffer containing the appropriate concentration of the compound to be tested to 10 l of TS2 buffer containing NAD+ , 30 nCi of [1,2,6,7-3 H]-cortisol and unlabeled cortisol to give a nal concentration of 400 M NAD+ and 10 nM cortisol. Stock solutions of the compounds in methanol or in DMSO were diluted in TS2 buffer to yield the appropriate concentrations, whereby the concentration of methanol or DMSO

2. Materials and methods 2.1. Materials Cell culture media were purchased from Invitrogen, Basel, Switzerland. [1,2,6,7-3 H]-cortisol was from Amersham Pharmacia, Dbendorf, Switzerland. [1,2,6,7-3 H]-cortisone was produced by incubating 1 mCi of [1,2,6,7-3 H]-cortisol

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in the reactions were below 0.1%. Control reactions with or without 0.1% of the solvent showed the same activity. After incubation at 37 C for 10 min with shaking, 10 l of stop solution containing 2 mM of unlabeled cortisol and cortisone dissolved in methanol were added. Conversion of radiolabeled cortisol was determined by separation of cortisol and cortisone using TLC and a solvent system of 9:1 (v/v) chloroform:methanol, followed by scintillation counting (Escher et al., 1997). In absence of inhibitors approximately 40% of cortisol was converted to cortisone. Similarly, reductase activity was measured in a reaction containing NADPH, 30 nCi of [1,2,6,7-3 H]-cortisone and unlabeled cortisone, whereby nal concentrations were 400 M NADPH and 10 nM cortisone. No loss of 11 -HSD2 activity was observed upon freezing of cell lysates for up to 1 month. In contrast, 11 -HSD1 activity declined after cell disruption, with a concomitant loss of afnity for its substrate but without any signicant loss of apparent Vmax . Activities were determined measuring the conversion of either radiolabeled cortisone or cortisol for 520 min using substrate concentrations in the range between 10 and 10 M. Analysis by EadieHofstee plots yielded a linear curve for the activities, indicating that the enzyme followed rst order rate kinetics in the range of substrate applied (Fig. 2). Apparent Km values, calculated assuming rst order rate kinetics, increased from 329 nM to 3.2 M for the reduction reaction and from 383 nM to 2.9 M for the oxidation reaction without signicant changes in apparent Vmax when reactions were carried out with fresh lysate or after incubation of the lysate on ice for 2 h before assessing enzyme activities. Therefore, 11 -HSD1 activities were measured immediately after cell disruption. All measurements included a negative control in absence of environmental compound and a positive control containing glycyrrhetinic acid at a nal concentration of

10 M. Results are expressed as mean S.D. and consist of at least four independent measurements. 11 -HSD1 activity in intact stably transfected HEK-293 cells was determined by distributing 100,000 cells per well of a 96-well plate and growth for 24 h, before addition of inhibitors at a nal concentration between 0 and 200 M for 10 min and 10 nM of radiolabeled cortisol or cortisone. Reactions were stopped by adding 2 mM unlabeled cortisol and cortisone dissolved in methanol after incubation for 1 h at 37 C and analyzed by TLC and scintillation counting. No conversion was observed when using untransfected HEK-293 cells and conversion was 2530% when using stably transfected cells in absence of inhibitors (negative control). 3T3-L1 preadipocytes were cultured in DMEM with 10% FCS and allowed to achieve conuence (day 2). Following medium replacement and incubation for another 2 days, differentiation was induced by adding 1 g/ml insulin, 0.5 M dexamethasone and 0.25 mM 3-isobutyl-1-methylxanthine (day 0). After 48 h the medium was replaced with DMEM containing 10% FCS and 1 g/ml insulin (day 2), and 3 days later (day 5) with DMEM containing 10% FCS. 11 -HSD1 activity in intact differentiated 3T3-L1 cells was determined at day 7 by adding radiolabeled cortisone at a nal concentration of 10 nM. After incubation of cells for 4 h at 37 C in presence or absence of avanoids, the amount of converted cortisone was assessed by ethylacetate extraction of steroids from the growth medium, separation by TLC and scintillation counting. 2.4. MTT cytotoxicity assay To avoid cell detachment, HEK-293 cells stably transfected with 11 -HSD1 or 11 -HSD2 were grown in

Fig. 2. 11 -HSD activities. Enzymatic activities of lysates from HEK-293 cells stably transfected with 11 -HSD1 (A and B) or 11 -HSD2 (C) were determined by measuring the reduction of cortisone to cortisol (A) or the oxidation of cortisol to cortisone (B and C) as described under Section 2. The insets show EadieHofstee plots of the concentration-dependent reduction of cortisone (A) or oxidation of cortisol (B and C). Representative experiments are shown.

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poly-d-lysine coated 96-well plates at 37 C. Cytotoxicity of tested compounds was monitored up to 200 M. The chemicals were added and cells were incubated for 4 h at 37 C, followed by a change to fresh medium containing 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After conversion of MTT, the medium was removed and 200 l of DMSO was added to the insoluble fraction. Conversion of MTT was kept below OD 0.9 (A570 A690 ). Chloroform (5%, v/v) and 50 mM sodium hydroxide were used as controls.

3. Results and discussion 3.1. Inhibition of human 11-HSD2 activity by environmental chemicals By protecting the MR from inappropriate activation by cortisol, 11 -HSD2 renders specicity to MR for aldosterone which regulates sodium balance and is essential for blood pressure control. It was demonstrated that glycyrrhetinic acid, a compound of licorice, is a potent inhibitor of 11 -HSD2, and that the excessive consumption of sweets containing glycyrrhetinic acid results in sodium retention causing increased blood pressure (Ferrari et al., 2001; Gomez-Sanchez and Gomez-Sanchez, 1992; Lindsay et al., 1996). Thus, the prolonged exposure to environmental chemicals that inhibit 11 -HSD2 are expected to result in increased intracellular cortisol concentrations that may lead to MR-mediated sodium retention and pose a risk factor for the development of hypertension. In the present study, several environmental chemicals including dietary phyto-compounds, herbicides, fungicides, pesticides, plasticizers and PCBs were tested for their ability to inhibit 11 -HSD2. For this purpose we measured 11 -HSD activites in lysates from stably transfected cells in a 96-well plate assay, allowing the rapid identication of 11 -HSD inhibitors. Lysates from HEK-293 cells stably transfected with 11 -HSD2 oxidized cortisol with an apparent Km of 195 16 nM and a Vmax of 1.3 0.2 nmol/h mg total protein (n = 4)(Fig. 2), compared with transiently transfected cells with a Km of 200 29 nM and a Vmax 2.9 0.4 nmol/h mg. The use of sub-Km concentrations of substrate may lead to some false negative results for compounds that exert weak inhibitory effects on 11 -HSD activity. To compare the inhibitory potency of the chemicals tested, glycyrrhetinic acid served as a positive control. The results of 11 -HSD inhibition studies are shown in Table 1. No activity was detected using untransfected cells. The widely used pesticide endosulfan, which has been shown to interfere with glucocorticoid metabolism by disrupting sites downstream of the cAMP-generating step of the cortisol synthetic pathway (Bisson and Hontela, 2002), inhibited 11 -HSD2 with an IC50 of 61 22 M. Inhibition of 11 -HSD2 was also observed for 4-tert-octylphenol and 4-nonylphenol with IC50 of 3023 and 7920 M, respec-

tively. The length of the aliphatic side chain played a critical role in the inhibitory potency of these chemicals, since 4-tert-butylphenol did not inhibit 11 -HSD2 at the concentrations applied. These chemicals are used as raw materials for detergents, emulsiers, anti-oxidants, pesticide and in PVC. Bisphenol A, which is a monomer for the synthesis of polycarbonate plastics, epoxy resins and composites and is used in packaging as coating in food cans, inhibited 11 -HSD2 with an IC50 of 50 19 M. Zearalenone, which is contained in mold-infected food, had an IC50 of 107 36 M. All of these compounds were weak inhibitors of 11 -HSD2. Zearalenone, endosulfan, 4-tert-octylphenol and 4-nonylphenol inhibited 11 -HSD2 at concentrations where cytotoxic effects were observed (Table 1). Although cytotoxic effects observed in HEK-293 cells may be different from that of cells in a given tissue and an accumulation of some of these chemicals in certain cells cannot be excluded, it is unlikely that such high intracellular concentrations are reached in vivo. The most potent inhibitor was diethyldithiocarbamate with an IC50 of 6.3 3.8 M, well below the cytotoxic concentration. Diethyldithiocarbamate belongs to the class of thiocarbamate chemicals which are widely used in agriculture as pesticides or fungicides but that are also used as vulcanization accelerators in latex production (mainly in gloves). Recently, the use of the potent anti-oxidant diethyldithiocarbamate as a short-term drug for acute hepatitis was suggested (DiSilvestro et al., 2002). However, the inhibition of copper metalloenzymes and the interference of diethyldithiocarbamate with glucocorticoid metabolism by inhibiting 11 -HSD2 have to be taken into consideration for a potential therapeutic application. All of the above mentioned chemicals inhibited 11 -HSD2 and may thus lead to increased intracellular glucocorticoid concentrations. 11 -HSD2 exerts an essential function in the placenta by protecting the fetus from excessive cortisol concentrations (Sun et al., 1998). The overexposure of a fetus to glucocorticoids leads to intrauterine growth retardation and disturbed hypothalamic-pituitary adrenal axis with a higher risk for the development of cardiovascular disease in adulthood (Bertram et al., 2001; Lesage et al., 2001). Abietic acid (Fig. 3), which is found in cosmetics such as foundations, concealers, sunscreens, mascaras, eyeliners, lipsticks, creams and color pencils that contain colophony, was shown to inhibit gastric secretions, suggesting its application in ulcer treatment (San Feliciano et al., 1993). Here, we demonstrate that abietic acid efciently inhibits 11 -HSD2 with an IC50 of 12 2 M. However, in contrast to the compounds above, abietic acid also efciently inhibited the oxidation of cortisol (IC50 2.8 0.8 M) and the reduction of cortisone (IC50 27 3 M) catalyzed by 11 -HSD1. Previous evidence indicated that abietic acid exerts in vivo anti-inammatory activity after oral or topical administration (Fernandez et al., 2001). Whether abietic acid leads to locally enhanced concentrations of cortisol due to an inhibition of 11 -HSD2 is not clear. Moreover, abietic acid partially inhibited the production of prostaglandin E2 in

R.A.S. Schweizer et al. / Molecular and Cellular Endocrinology 212 (2003) 4149 Table 1 Inhibition of human 11 -HSD enzyme activities by environmental compounds ( M) Compound Glycyrrhetinic acida Flavanone compounds Flavanone 6-Hydroxyavanone 4 -Hydroxyavanone 2 -Hydroxyavanone Naringenin Hesperetin Flavone compounds Flavone 7-Hydroxyavone Apigenin Chrysin Kaempferol Quercetin Isoavone compounds Biochanin A Genistein Daidzein Coumestrol Resorcinol Other natural compounds -Sitosterol Abietic acid Methyljasmonate Zearalenone Nicotin Fusidic acid Phtalates Di(2-ethylhexyl)phtalate, DEHP Diethylphtalate, DEP Di-n-butylphtalate, DBP Diisononylphtalate, DINP Butylbenzylphtalate, BBP Other environmental chemicals 4-tert-Octylphenol 4-tert-Butylphenol 4-Nonylphenol Bisphenol A Decabromdiphenylether, DecaBDE 2,2 -Dihyhroxybiphenyl 4,4 -Dihyhroxybiphenyl 4,4 -DDE Vinclozolin Atrazine Nitrofen 2-Naphthol Endosulfan Dibenzoylmethane Diethyldithiocarbamate, DETC 11 -HSD1 Reduction (IC50 ) 1.57 0.43 18 7.3 187 23 34 3.3 10 5.6
c c

45

11 -HSD1 Oxidation (IC50 ) 0.034 0.005

b c b b b c

11 -HSD2 Oxidation (IC50 ) 0.031 0.007

c b c c c c

Cytotoxicity n.d. >200 >100 >200 >200 >200 >200 >200 >50 >200 >50 >200 >200 >100 >200 >20 >20 >200 n.d. >200 >200 >50 >200 >70 >200 >200 >200 >200 >200 >20 >150 >25 >200 >15 >200 n.d. >50 >200 >200 >200 >200 >50 >50 >40

b b b c c b

c b c b b c

c b c b c c

b b b b b

b b b b b

b c b b c

27 3 107 35
c b c

2.8 0.8
c

12 2
c

84 9
b b

107 36
c

134 5
b c c c c

b b b c c

b b b b c

c b c b b c b b b b b c b

85 15
b

30 23
c

178 22
c b

79 20 50 19
b b b c c c c c

151 31
b b c b b b c c b

52 17
b

61 22
c

6.3 3.8

IC50 values were obtained by graphical and mathematical determinations. Values represent mean S.D. of at least four independent experiments. n.d., not determined. a Used as controls. b No inhibition detectable. c IC 50 > 200 M.

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Fig. 3. Chemical structures of avone, avanone, naringenin and abietic acid.

lipopolysaccharide treated macrophages (Fernandez et al., 2001). Other investigators demonstrated that the induction of 11 -HSD1 potentiated the subsequent stimulation of prostaglandin H synthetase 2 expression and secretion of prostaglandin E2 by cortisone in chorionic trophoblast (Sun et al., 2002). Thus, the reduced secretion of prostaglandin E2 upon administration of abietic acid may be explained by the inhibition of 11 -HSD1. On the other hand, inhibition of 11 -HSD1 would result in a lower concentration of the immunosuppressive cortisol. 3.2. Inhibition of human 11-HSD1 activity by environmental chemicals There is increasing evidence that the nutritional and hormonal environment encountered by the fetus affects fetal growth and the risk for cardiovascular disease (Godfrey and Barker, 2000; Sun et al., 1998). Enhanced glucocorticoid levels lead to increased gluconeogenesis and antagonize the metabolic actions of insulin. In addition, glucocorticoids regulate fetal growth and development. 11 -HSD1 plays an essential role in the local activation of the GR by converting the inactive cortisone to the biologically active cortisol (Stewart and Krozowski, 1999; White et al., 1997). Overexpression of 11 -HSD1 in transgenic mice caused central obesity and resulted in metabolic syndrome and diabetes type 2 (Masuzaki et al., 2001). Recently, the use of selective 11 -HSD1 inhibitors in the treatment of diabetes type 2 was suggested and a new class of arylsulfonamidothiazoles were shown to selectively inhibit 11 -HSD1 and to reduce blood glucose levels in diabetic mice (Alberts et al., 2002; Barf et al., 2002). Thus, compounds selectively inhibiting 11 -HSD1 may have a positive effect in individuals with metabolic syndrome and diabetes type 2. The frequent consumption of fruits and vegetables has a positive impact on human health. Many of these effects are exerted by phytoestrogens which were shown to lower serum cholesterol

(Anderson et al., 1995), act as anti-oxidants and free radical scavengers (Jha et al., 1985) and reduce cancer risk (Messina et al., 1994). Lysates from HEK-293 cells stably transfected with 11 -HSD1 oxidized cortisol with an apparent Km of 383 8 nM and a Vmax of 1.6 0.2 nmol/h mg total protein (n = 3) and reduced cortisone with an apparent Km of 329 43 nM and a Vmax of 1.4 0.3 nmol/h mg total protein (Fig. 2), compared with transiently transfected cells with a Km of 453 10 nM and a Vmax 1.9 0.1 nmol/h mg for oxidation and a Km of 344 32 nM and a Vmax of 1.4 0.2 nmol/h mg total protein for the reduction. No 11 -HSD1 activity was detected using untransfected cells. Zearalenone, 4-tert-octylphenol, 4-tert-nonylphenol and 2,2 -dihydroxybiphenyl inhibited the oxidative activity of 11 -HSD1 but not its reductive activity. Since 11 -HSD1 acts predominantly as a reductase in vivo, it is unlikely that the local activation of GR by glucocorticoids would be affected by these compounds due to inhibition of 11 -HSD1 in vivo, these compounds may, however, lead to enhanced 11 -hydroxyglucocorticoid levels by inhibiting 11 -HSD2. In contrast, methyljasmonate and dibenzoylmethane selectively inhibited the reduction by 11 -HSD1, although at relatively high concentrations. Furthermore, our results demonstrate that avanone (Fig. 3) and the monohydroxylated avonoids 2 -hydroxyand 4 -hydroxyavanone are potent inhibitors of the reductive activity of 11 -HSD1 with IC50 in the low micromolar range (Table 1, Fig. 4). None of the avonone derivatives tested showed cytotoxic effects at concentrations below 100 M. When measured in stably transfected HEK-293 cells, avanone selectively inhibited the reductive activity of 11 -HSD1 (IC50 18 M), and no inhibition of the oxidative activity of either 11 -HSD1 or 11 -HSD2 was observed at concentrations below 200 M. Flavanone also efciently inhibited the reduction of cortisone by 11 -HSD1 expressed in intact HEK-293 cells with an IC50 of 8.7 2.2 M, suggesting that similar concentrations may be effective in vivo. The IC50 for 2 -hydroxyavanone using intact cells was 57 24 M. The reason for the higher concentrations of 2 -hydroxyavanone required to inhibit 11 -HSD1 activity in experiments using intact cells (IC50 57 24 M) versus experiments with lysates (IC50 10 5.6 M) when compared with the values obtained for avanone (IC50 8.7 2.2 and 18 7.3) is not clear. The inhibition of 11 -HSD1 expressed endogenously in differentiated 3T3-L1 adipocytes by avanone (IC50 13 1 M) and naringenin (IC50 364 30 M) was comparable with the data obtained from transfected HEK-293 cells (Fig. 5). In contrast to the less inhibitory effect on 11 -HSD1 in HEK-293 cells, 2 -hydroxyavanone efciently inhibited 11 -HSD1 in 3T3-L1 adipocytes (IC50 1.8 0.5 M). These ndings suggest more efcient uptake of 2 -hydroxyavanone by 3T3-L1 adipocytes than by HEK-293 cells. Whether specic uptake by 3T3-L1

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Fig. 4. Inhibition of 11 -HSD1-dependent reduction of cortisone by avonoids. Lysates were prepared from HEK-293 cells stably transfected with 11 -HSD1. Activities were determined immediately after disruption of cells. The inhibitory effect of avonoids on the conversion of cortisone (10 nM) to cortisol was determined. The activity is given as percentage of control, e.g. activity in absence of avonoids. IC50 values were calculated from four independent experiments (mean S.D.).

adipocytes or extrusion by HEK-293 cells is responsible for the observed difference remains to be elucidated. Determination of the reduction of various concentrations of cortisone by 11 -HSD1 in presence or absence of 25 M avanone revealed an increase of the apparent Km for cortisone from 32943 nM to 70448 nM without an alteration

of Vmax (1.4 0.2 nmol/h mg total protein) upon the addition of avanone. These experiments suggest that avanone may act as a competitive inhibitor of 11 -HSD1. The circulating concentrations of total phytoestrogens in the Japanese diet ranges from 1 to 5 M (Adlercreutz et al., 1993; Morton et al., 1994). Infants fed with soy-based

Fig. 5. Flavanoid-dependent inhibition of the reduction of cortisone in intact differentiated 3T3-L1 adipocytes. Intact differentiated 3T3-L1 adipocytes were incubated with 10 nM radiolabeled cortisone and various concentrations of avanoids. Activities were determined as described in Section 2 and are given as percentage of control, e.g. activity in absence of avonoids. IC50 values were calculated from four independent experiments (mean S.D.).

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formula contain about 4 M of genistein and daidzein in their plasma (Setchell et al., 1997). Thus, it is likely that the concentrations of avanoids required to inhibit 11 -HSD1 are reached in these individuals. 3.3. Structural requirements for avonoids inhibiting 11-HSD1 In contrast to avanone and the monohydroxylated derivatives 2 -hydroxy- and 4 -hydroxyavanone, hydroxylation at position 6 or multiple hydroxylation of avanone dramatically reduced its inhibitory effect on 11 -HSD1. Naringenin, containing three hydroxyl groups in positions 7, 5 and 4 , was a weak inhibitor with an IC50 >200 M (Figs. 3-5). The physiological effect of naringenin, reported previously to inhibit 11 -HSD1 (Hult et al., 1998) and 11 -HSD2 (Reidenberg, 2000), is unclear. Although it was suggested that the inhibition of 11 -HSD2 by naringenin may explain the observed reduced cortisone/cortisol ratio in individuals drinking 2 l of grapefruit juice daily, it is unlikely that concentrations close to the reported IC50 of 300350 M (Hult et al., 1998; Reidenberg, 2000) are reached in vivo. These data rather suggest that a yet unidentied compound potently inhibiting 11 -HSD2 is present in grapefruit juice. In contrast to avanone, avone and its derivatives showed very low or no inhibitory effect on both 11 -HSD1 and 11 -HSD2 activity. The double bond between atoms C2 and C3 reduced the inhibitory potential of these molecules. This is in clear contrast to 17 -HSD5 which is inhibited potently by avone derivatives with an increasing inhibitor potency with increasing number of hydroxylations (Krazeisen et al., 2001). 3.4. Specicity of avanone for 11-HSD1 and modulation of other enzymes involved in steroid hormone metabolism It was previously reported that avanone is a weak inhibitor of 17 -HSD5 (IC50 50 M) and aromatase (IC50 14 M) but did not inhibit human placental 17 -HSD activity (Le Bail et al., 2001). Hydroxylated avone derivatives were shown to interfere with glucocorticoid biosynthesis, whereas there is no evidence for a similar effect of avanone and its derivatives. Among others, 6-hydroxyavone was demonstrated to inhibit 3 -HSD, P450c17 and P450c21 which are essential for the conversion of pregnenolone to progesterone, 17-hydroxyprogesterone and 11-deoxycortisol (Ohno et al., 2002). Moreover, a decreased cortisol production in human adrenal cells, which also express 11 -HSD1, was observed for 6-hydroxyavone (6% of control at 12.5 M). Thus, a soy-based diet containing both avone and avanone compounds may result in reduced glucocorticoid concentrations due to avone compound-dependent inhibition of glucocorticoid biosynthesis and avanone compound-dependent inhibition of 11 -HSD1. In conclusion, our results suggest that avanone

may be used in the treatment of individuals with metabolic syndrome or diabetes type 2. Moreover, avanone might be subjected to a lead optimization process in order to synthesize novel and more potent selective 11 -HSD1 inhibitors. Our study may explain some of the benecial effects of red and yellow fruits and vegetables, containing avanone and its derivatives, on body weight control. Acknowledgements We thank Jaqueline Kaempf for expert technical support in the construction of stably transfected cells and Dr. Galina Apostolova for advice in 3T3-L1 differentiation and activity assay. This work was supported by a grant from the Swiss National Science Foundation NRP50 Endocrine Disruptors No. 4050-066575. References
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