Eas Al-Dujaili
email: ealdujaili@qmu.ac.uk

C J Kenyon

M R Nicol

J I Mason

Dr Emad Al-Dujaili

Liquorice and glycyrrhetinic acid increase DHEA and deoxycorticosterone levels in vivo and in vitro by inhibiting adrenal SULT2A1 activity

A c c e p t e d M a n u s c r i p t

ABSTRACT

The mineralocorticoid effects of liquorice are mediated by the inhibitory effects of one of its active components glycyrrhetinic acid on 11β-hydroxysteroid dehydrogenase type 2.

However, liquorice is reputed to have many medicinal properties and also contains a number of other potentially biologically active compounds. Here we have investigated the wider effects of oral liquorice on steroidogenesis focussing particularly on possible inhibitory effects of glycyrrhetinic acid on adrenal sulfotransferase activity. Salivary steroids were profiled by ELISA in groups of normal male and female volunteers after consuming either liquorice-containing or non-liquorice-containing confectionary for one week. Cortisol and cortisone levels reflected expected inhibition of 11β-hydroxysteroid dehydrogenase type 2 by glycyrrhetinic acid. Salivary aldosterone was decreased but deoxycorticosterone, dehydroepiandrosterone and testosterone were increased. To assess whether glycyrrhetinic acid directly affected steroidogenesis, free and conjugated steroids were measured in incubates of adrenocortical H295 cells, firstly, in the presence or absence of forskolin and, secondly, with radiolabeled deoxycorticosterone or dehydroepiandrosterone. Glycyrrhetinic acid inhibited cortisone and enhanced cortisol synthesis consistent with 11βhydroxysteroid dehydrogenase type 2 inhibition. Basal and forskolin-stimulated synthesis of deoxycorticosterone and dehydroepiandrosterone conjugates were also inhibited in a dosedependent manner; glycyrrhetinic acid inhibited the conjugation of deoxycorticosterone and dehydroepiandrosterone with IC50 values of 7µM. Inhibition of deoxycorticosterone and dehydroepiandrosterone conjugation was apparent within 4h of starting glycyrrhetinic acid treatment and was not associated with changes in the expression of SULT mRNA. SULT2A1 encodes the enzyme sulfotransferase 2A1 which is responsible for the sulfonation of deoxycorticosterone and dehydroepiandrosterone as well as pregnenolone and 17-A c c e p t e d M a n u s c r i p t 3 hydroxypregnenolone in human adrenal glands. We suggest that the glycyrrhetinic acid constituent of liquorice increases circulating and, thereby, salivary levels of unconjugated deoxycorticosterone and dehydroepiandrosterone by inhibiting their conjugation at source within the adrenal cortex. This effect may contribute to the mineralocorticoid actions of glycyrrhetinic acid and gives substance to claims that liquorice also has androgenic

Introduction

The hypertensive effects of liquorice and its active constituent glycyrrhetinic acid (GA) have been widely investigated [START_REF] Conn | Licorice-induced pseudoaldosteronism. Hypertension, hypokalemia, aldosteronopenia, and suppressed plasma renin activity[END_REF][START_REF] Farese | Licoriceinduced hypermineralocorticoidism[END_REF], and attributed mainly to competitive inhibition of 11β-hydroxysteroid dehydrogenase type 2 (11HSD2) [START_REF] Walker | Licorice-induced hypertension and syndromes of apparent mineralocorticoid excess[END_REF]. This enzyme is expressed in mineralocorticoid target tissues (eg colon and renal collecting duct) and normally serves to prevent glucocorticoid hormones from occupying mineralocorticoid receptors by converting biologically active cortisol to inactive cortisone. When 11HSD2 is inhibited, endogenous glucocorticoids act as mineralocorticoid hormones. However, liquorice root has been used medicinally for many years to treat a variety of conditions [eg dry cough, asthma, thirst, sterilility, fever, digestive problems [START_REF] Davis | Medicinal uses of licorice through the millennia: the good and plenty of it[END_REF][START_REF] Hussain | The sweet cake that reaches parts other cakes can't![END_REF]] and, more recently by the body building fraternity, to increase muscle mass (www.bodybuilder.com/store/now/lic/html; www.crticalbench.com/licorice-root-supplement-review.htm). These properties are not necessarily mediated by GA and could be due to one or more of the many other biologically active constituents of liquorice root (eg flavonoids, isoflavonoids, polysaccharides, coumarins, sterol, phytoestrogens, volatile oils, glycyramarine as well as vitamins, minerals and amino acids [START_REF] Asl | Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds[END_REF][START_REF] Lee | Rapid identification and preparative isolation of antioxidant components in licorice[END_REF][START_REF] Shibata | A drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice[END_REF].

In addition to effects on glucocorticoid metabolism GA is thought to have other endocrine effects. For example, there have been several reports [START_REF] Armanini | Reduction of serum testosterone in men by licorice[END_REF][START_REF] Takahashi | Effect of a traditional herbal medicine (shakuyaku-kanzo-to) on testosterone secretion in patients with polycystic ovary syndrome detected by ultrasound[END_REF][START_REF] Takeuchi | Effect of paeoniflorin, glycyrrhizin and glycyrrhetic acid on ovarian androgen production[END_REF] albeit disputed [START_REF] Josephs | Liquorice consumption and salivary testosterone concentrations[END_REF] that GA and glycyrrhyzic acid (GZ) inhibit 17-20-lyase and 17-hydroxysteroid dehydrogenase activities thereby reducing testosterone synthesis in males and females by blocking the conversion of 17hydroxyprogesterone (17-OHP) to androstenedione and then the conversion of androstenedione to testosterone.

A c c e p t e d M a n u s c r i p t 5 Here we have gained a more comprehensive view of the effects of liquorice and GA on adrenal steroidogenesis by assaying mineralocorticoids, glucocorticoids and androgens in vivo in saliva of normal volunteers. We and others have found salivary steroid levels to be useful indicators of circulating plasma concentrations. [START_REF] Atherden | Development and validation of a direct radioimmunoassay for aldosterone in saliva[END_REF][START_REF] Conway | Raised salivary testosterone in women is associated with increase attraction to masculine faces[END_REF][START_REF] Groschl | The physiological role of hormones in saliva[END_REF]. Samples can be readily obtained non-invasively from patients and volunteers without the need of supervision. The effects of GA on corticosteroidogenesis have been modelled using a multipotent human adrenocortical cell line (H295) [START_REF] Rainey | Adrenocortical cell lines[END_REF]. These cells secrete aldosterone, cortisol and DHEA in response to a variety of trophic factors although responsiveness to ACTH is impaired due to an abnormal melanocortin receptor. This problem is bipassed by using forskolin to raise levels of cyclic AMP.. Based on our preliminary findings we have developed a hypothesis that GA influences circulating levels of adrenocortical derived steroid hormones by inhibiting their conjugation. In particular we have focussed on the hydroxysteroid sulfotransferase SULT2A1, an enzyme responsible for the sulfonation of DHEA, DOC and other steroids which are expressed in human adrenocortical tissue and H295 cells [START_REF] Rainey | Dissecting human adrenal androgen production[END_REF][START_REF] Strott | Sulfonation and molecular action[END_REF][START_REF] Kumar | Effect of insulin and testosterone on androgen production and transcription of SULT2A1 in the NCI-H295R adrenocortical cell line[END_REF].

Materials and Methods

Human study

The effects of dietary liquorice on salivary steroid hormone levels were tested in groups of 10 males and 10 female volunteers in a study that was approved by Queen Margaret University's Ethical Committee. Exclusion criteria were high (systolic >140mmHg) or low (diastolic < 60mmHg) blood pressure, treatment with drugs affecting blood pressure or endocrine functions (including oral contraceptives), menstrual disorders, allergy to liquorice, and a history of kidney or liver disease.

The study followed a cross-over design to compare the effects of liquorice-containing confectionery (contents: wheat flour, glucose syrup, modified maize starch, 3% liquorice extract, caramel, salt, caramel flavouring) and non-liquorice confectionery of the same sugar content and calorific value (contents: sugar, glucose syrup, water, maize starch, beef gelatine, flavourings, cochineal colour). Volunteers were asked to consume daily 100g of nonliquorice confectionery for 1 week, followed by a wash period of 3 days, and then the liquorice confectionary for another week. Female participants were tested during the follicular phase of the menstrual cycle with day1 of the test corresponding to the start of the cycle. Body weight, height and blood pressure when lying down were recorded before and at the end of each arm of the study. Mean participant age was 23.12 ± 5.6 years and BMI was 23.26 ± 4.37 kg/m 2 . There was no significant change in BMI with either arm of the study.

Participants were given very detailed instructions on how to collect and store saliva samples.

On days 7 and 10 of treatment, participants provided three saliva samples: (i) 30 minutes after waking (8.00 -9.00h); (ii) mid-morning between 11 and 13.00h; (iii) in the evening between 19.00 and 20.00h.

A c c e p t e d M a n u s c r i p t 7

Salivary steroids

Coded saliva samples were stored at -20°C prior to assay for cortisol, cortisone, aldosterone, deoxycorticosterone (DOC), testosterone and dehydroepiandrosterone (DHEA).

Steroids were extracted from saliva samples with diethyl ether and measured using sensitive and specific ELISAs modified from methods previously described [START_REF] Al-Dujaili | Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine[END_REF]Al-Dujaili et al., 2009a;Al-Dujaili et al., 2009b). Briefly, 0.5mL of saliva was vortexed with 5mL diethylether for 10min then placed in a -80C freezer. The ether was then decanted from the frozen aqueous layer into a glass tube and evaporated to dryness under nitrogen. The residue was reconstituted in 0.5mL of assay buffer. Free and conjugated levels of DHEA, DOC and other steroids were measured separately in pooled 0.5mL aliquots of male saliva after fractionation using C18 Sep-Pak cartridges (Waters Ltd, Elstree, Hertfordshire, UK).

Conjugated steroids were hydrolysed prior to assay by incubation with extracts of Helix pomatia, then extracted with ether and assayed.

In vitro studies

NCI-H295 cells (CRL-2128, ATCC, Rockville, MD, USA) were cultured in Dulbecco's modified Eagle's medium /F12 (DMEM/F12, Invitrogen, Paisley, Renfrew, UK) supplemented with 2% cosmic calf serum (Hyclone, Thermo Scientific, Cramlington, UK), 5 μg/mL insulin, 5 μg/mL transferrin and 5 ng/mL sodium selenite (as 1% ITS, Invitrogen) at 37°C with 5% CO 2 -95% air. Cells were seeded at 500,000 cells/well in 6-well plates. For experiments, cells were serum-deprived overnight and incubated for up to 24h with various concentrations of glycyrrhetinic acid (0-50μM) in the presence or absence of forskolin (10 mM). In some experiments steroid conjugation was measured directly by incubating cells Solon, Ohio, USA). The medium was removed and stored at -80°C for steroid analysis; remaining cells were lyzed and RNA extracted using RNeasy mini-kits (Qiagen, Crawley, West Sussex, UK). Steroids were extracted from the medium using C18 Sep-Pak cartridges; conjugated and free steroids were eluted separately with 40% and 100% aqueous methanol respectively. Radioactivity in free and conjugated fractions was measured by scintillation counting. Absolute levels of steroids were measured by ELISA; conjugated steroids were hydrolysed with Helix pomatia prior to assay.

Real time PCR assays

RNA was extracted using Qiagen RNeasy Mini kit and genomic DNA was excluded by DNase treatment of samples, on-column, with the RNase free DNase set (Qiagen) according to supplier's protocol. RNA purity and quantification were assessed spectrophotometrically (Nanodrop model ND-1000, Nanodrop Technologies Inc, Wilmington, DE, USA). mRNA (200ng) was reversed transcribed using RT-Reagent kits (Applied Biosystems, Applera, Warrington, Cheshire, UK). . Real-Time PCR was performed for SULT2A1 cDNA and ribosomal 18S RNA using pre-validated Assay-on-Demand System reagents (Applied Biosystems). Briefly, 2μL cDNA was used as a template mixed with 1X universal Taqman master cocktail (Applied Biosystems) and the specific set of 1X primer/probe mixture.

Duplicate reactions in 96-well plates were evaluated using an ABI Prism 7900 Sequence Detector (Applied Biosystems). The C T value obtained for the 18S species was used to confirm cDNA quality and as an internal reference control for SULT2A1 expression. 18S RNA has been used routinely in our experiments with H295 cells over several years and has proved to be very reliable for quality control and normalisation purposes. For salivary steroids the sex-dependent and daytime variations of the effects of liquorice containing and non-liquorice containing confectionery were compared using a general linear model of analysis of variance. Data for the effects of confectionery were matched for each individual. For studies of steroid synthesis and SULT2A1 expression in H295 cells, the effects of forskolin and GA were tested by analysis of variance with Neuman-Keul's correction. Results are expressed as mean values ± SE and P < 0.05 was considered statistically significant.

Results

Effects of liquorice on salivary steroid concentration

There was no significant increase in systolic or diastolic blood pressure due to 1 week consumption of liquorice confectionery. Figure 1 and Table 1 shows that the overall effect of liquorice on salivary steroid concentrations in males and females is broadly similar. Cortisol, deoxycorticosterone, DHEA and testosterone were increased (although not significantly for cortisol in males) whereas aldosterone and cortisone were decreased. Effects on cortisol and cortisone are in line with known inhibition of 11β-hydroxysteroid dehydrogenase by liquorice and GA which are known to cause hypertension via a mineralocorticoid-dependent process.

Apparent mineralocorticoid excess due to cortisol occupancy of MR is reflected by decreased aldosterone levels.

Increased DHEA and testosterone levels in response to liquorice were not expected. These effects are more prominent in the evening when cortisone levels are least affected (figure 2).

Potentially testosterone increases could arise from peripheral conversion of DHEA derived from the adrenal gland. However, salivary testosterone and DHEA concentrations were Table 2 shows the effects of liquorice on free and conjugated steroids in pooled saliva from male subjects. The pattern of fractionated free steroid values was similar to that of unfractionated steroid values. Levels of DHEA and DOC conjugates were decreased by liquorice treatment; other steroid conjugates were not significantly affected.

Effects of GA on basal and forskolin-stimulated steroidogenesis by H295 cells

Responsiveness to ACTH is compromised in H295 cells although cyclic AMP-dependent signalling is preserved. Forskolin stimulates adenyl cyclase independent of ACTH receptors and was used in the present studies to stimulate steroidogenesis via cyclic AMP. Figure 3 shows that synthesis of unconjugated steroids were all stimulated by forskolin (DHEA > cortisol> cortisone> deoxycorticosterone > aldosterone). Similar results were obtained with vasoactive intestinal peptide, an agonist with activity which is not dependent on cyclic AMP (data not shown). As expected of an adrenocortical cell line, very low levels of testosterone were detected in the medium and were unresponsive to forskolin treatment (data not shown).

GA reduced both basal and forskolin-stimulated cortisone synthesis (P<0.01) but did not significantly affect cortisol synthesis. Basal and forskolin-stimulated DOC and DHEA synthesis were increased by GA but only effects on basal synthesis were statistically significant (P<0.01). Aldosterone responses to GA were not statistically significant.

Dose dependent effects of GA on conjugation of steroids synthesized by H295 cells

In the absence of GA similar amounts of DHEA and DHEAS were released by H295 cells; the degree of conjugation was not affected by forskolin (figure 4). DOC conjugation was A c c e p t e d M a n u s c r i p t 11 approximately half that of DHEA and, in the absence of GA was slightly higher in forskolinstimulated cells. Cortisol, cortisone and aldosterone conjugation was 9.9, 30.0 and 62% respectively in unstimulated cells and was reduced by forskolin treatment (3.3, 15 and 45%) but not by GA either in the presence or absence of forskolin. GA caused dose-dependent decreases in the degree of DOC and DHEA conjugation in both basal and forskolinstimulated cells. Non-conjugated DHEA and especially DOC were increased by low doses of GA (≤ 10µM) commensurate with decreased conjugation. However, the total amounts of DHEA released (conjugated plus unconjugated) were inhibited by high doses of GA (≥ 25µM).

GA inhibits 11β-hydroxysteroid dehydrogenase activity in H295 cells

Cortisone was markedly inhibited by GA (figure 5). The ratio of cortisone:cortisol was decreased by the lowest dose of GA (1µM) in forskolin stimulated cells (P=0.01) indicating that 11β-hydroxysteroid dehydrogenase activity is more sensitive to the inhibitory effects of GA than enzymes responsible for conjugation. However, the net effect of GA on cortisol synthesis due to 11HSD2 inhibition is probably negligible since, in the absence of GA, the proportion of cortisol converted to cortisone is less than 3% for unstimulated cells and less than 1% for forskolin-stimulated cells.

GA inhibits conjugation of radiolabeled DOC and DHEA

To confirm that GA inhibits conjugation of DOC and DHEA rather than de novo synthesis of steroids, H295 cells were incubated with either 3 H-DHEA or 3 H-DOC.

Conjugation of both steroids was inhibited partially with 3µM GA and by more than 80% with 30µM GA (figure 6). The inhibitory effects of the higher dose of GA were apparent within 4h (figure 7).

GA effects on conjugation are not due to inhibition of SULT2A1 gene transcription

Previous studies have identified DOC and DHEA sulfates as conjugates that are derived principally from the adrenal gland due to the specific expression of SULT2A1. To establish whether GA effects on DOC and DHEA conjugation were due to inhibition of transcription, SULT2A1 mRNA transcript levels were measured by quantitative RT-PCR in cells treated with GA. Despite evidence of marked inhibition of steroid conjugation, SULT2A1 mRNA levels were unaffected (figure 7), suggesting that GA directly affects SULT2A1 enzyme activity.

Discussion

The effects of liquorice on salivary steroid levels are, in part, explained by the known inhibitory effects of glycyrrhetinic acid (GA) on 11β-hydroxysteroid dehydrogenase activity [START_REF] Conn | Licorice-induced pseudoaldosteronism. Hypertension, hypokalemia, aldosteronopenia, and suppressed plasma renin activity[END_REF][START_REF] Farese | Licoriceinduced hypermineralocorticoidism[END_REF][START_REF] Walker | Licorice-induced hypertension and syndromes of apparent mineralocorticoid excess[END_REF]. The hypertensinogenic response to liquorice is due to a block in the conversion of active endogenous glucocorticoid hormone, cortisol, to inactive cortisone. The isoform of the enzyme responsible for this conversion, 11β HSD2, is expressed in mineralocorticoid responsive target tissues where it serves to prevent high concentration of circulating glucocorticod hormones from binding inappropriately to mineralocorticoid receptors (MR). When 11HSD2 is inhibited, as shown by marked decrease in salivary cortisone:cortisol ratios, then glucocorticoids can cause MRdependent sodium retention, plasma volume expansion and ultimately an increase in blood pressure [START_REF] Quinkler | Hypertension and the cortisol-cortisone shuttle[END_REF][START_REF] Walker | Licorice-induced hypertension and syndromes of apparent mineralocorticoid excess[END_REF]. In the present study, liquorice did not cause hypertension perhaps because the period of treatment was too short or the dose of liquorice too small [START_REF] Sigurjonsdottir | Liquorice-induced rise in blood pressure: a linear dose-response relationship[END_REF]. However, there was a decrease A c c e p t e d M a n u s c r i p t 13 in salivary aldosterone, an indication of the suppression of the renin-angiotensin-aldosterone system (RAAS) when endogenous glucocorticoids activate MR. It should be noted that GA did not inhibit aldosterone synthesis by H295 cells indicating that aldosterone decreases in vivo are secondary to mineralocorticoid excess caused by other steroids. The ratio of cortisone:cortisol synthesised by H295 cells is low (<0.02) and was reduced even further by GA. Assuming that endogenous adrenocortical production similarly favours cortisol over cortisone, it seems likely that the much higher cortisone to cortisol ratios in saliva reflect the effects of GA on extra-adrenal glucocorticoid metabolism.

Liquorice also caused increases in salivary DOC, DHEA and testosterone which may have additional pharmacological consequences. Firstly DOC is recognised as a weak mineralocorticoid and could, therefore, contribute to hypertensinogenic processes and suppression of RAAS. Secondly, increased DHEA and testosterone could have androgenic effects. This observation supports claims that liquorice promotes increases in muscle mass (www.bodybuilder.com/store/now/lic/html; www.crticalbench.com/licorice-root-supplementreview.htm) . It should be noted, however, that others have reported that GA binds to the androgen receptor and could, therefore, exert more direct effects [START_REF] Tamaya | Inhibition by plant herb extracts of steroid bindings in uterus, liver and serum of the rabbit[END_REF].

The mechanism responsible for increases in DOC, DHEA and testosterone are not immediately apparent. None of them could be considered substrates for 11HSD2 although androgen and DOC metabolites are antagonists of 11HSD activity [START_REF] Morris | Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol[END_REF]. Three possible explanations are suggested: (i) GA has been shown to inhibit two hepatic enzymes (3β-hydroxysteroid dehydrogenase and steroid 5β reductase) responsible for ring A reduction of steroid hormones [START_REF] Latif | The effects of the licorice derivative, glycyrrhetinic acid, on hepatic 3-alpha-and 3-beta-hydroxysteroid dehydrogenases and 5-alpha-and 5-beta-reductase pathways of metabolism of aldosterone in male rats[END_REF]. This could prolong the plasma half life of DOC and testosterone. However, aldosterone and cortisone are also catabolised by this route yet are present at lower not higher concentrations in saliva after liquorice treatment; (ii) GA could affect de novo androgen biosynthesis. It has been suggested that 11HSD1 activity modulates A c c e p t e d M a n u s c r i p t 14 the inhibitory effects of glucocorticoids on testosterone synthesis by the Leydig cells of the testis [START_REF] Hu | Inhibition of 11beta-hydroxysteroid dehydrogenase enzymatic activities by glycyrrhetinic acid in cells[END_REF][START_REF] Monder | 11 beta-Hydroxysteroid dehydrogenase alleviates glucocorticoid-mediated inhibition of steroidogenesis in rat Leydig cells[END_REF]. If GA were to inhibit the capacity of 11HSD1 to regenerate active glucocorticoid hormone, this would lessen the effects of glucocorticoids on androgen production. Against this hypothesis is the fact that the 11HSD1 enzyme is less susceptible to GA inhibition than 11HSD2 [START_REF] Latif | Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5-alpha-reduced (but not 5-beta) derivatives of adrenocorticosteroids[END_REF]Marandici and Monder, 1993) and is thought to contribute to dehydrogenase activity in Leydig cells [START_REF] Ge | 11{beta}-Hydroxysteroid dehydrogenase 2 in rat Leydig cells: its role in blunting glucocorticoid action at physiological levels of substrate[END_REF]. Also in the present study, effects of liquorice on salivary androgens were seen in both males and females in the presence of higher cortisol levels; (iii) GA may directly affect adrenal androgen production. In primates, DHEA and its sulfate are derived predominantly from the adrenal gland with males producing slightly more than females [START_REF] Labrie | Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone[END_REF]. DHEA is converted to testosterone by peripheral tissues. To test this third possibility, we measured steroidogenesis in H295 cells treated with GA. H295 cells are a multipotent adrenal cell line which synthesise DHEA, DOC, cortisol and cortisone and aldosterone. A preliminary study confirmed that the profile of steroids by these cells in response to GA (figure 3) was similar to those of saliva after feeding liquorice except that testosterone levels were very low and unresponsive to trophic factors and did not respond consistently to GA. We were aware however, that GA induced changes reflected free steroid levels and did not take account of the very high levels of conjugated steroids that are synthesised by the adrenal cortex and by H295 cells. In particular, it is known that pregnenolone, 17-hydroxypregnenolone, deoxycorticosterone and DHEA are secreted as sulfates [START_REF] Rainey | Dissecting human adrenal androgen production[END_REF][START_REF] Strott | Sulfonation and molecular action[END_REF].

The effects of GA on conjugated and free steroid production were tested in basal and stimulated H295 cells. Conjugated DHEA and DOC were decreased in a dose dependent manner whereas free DHEA and DOC were increased by 10µM GA. In contrast, the increase in total cortisol caused by 10µM GA was related to inhibition of 11HSD activity since cortisone was decreased and cortisol conjugation was unaffected. High doses of GA (> A c c e p t e d M a n u s c r i p t 15 50μM) tended to reduce synthesis of total DHEA and increases in DOC and cortisol appeared to have reached a plateau. Arguably this high dose effect could be a non-specific toxic effect although there were no signs of cell death. An alternative possibility is that GA inhibits the sulfation of pregnenelone, a known substrate of adrenal sulfotransferase activity and an early intermediate in glucocorticoid biosynthesis. This would shunt steroidogenesis towards the synthesis of DOC and could also contribute to the amounts of basal and forskolin stimulated total cortisol release that were increased by GA (figure 5).

Previous studies have identified DOC and DHEA conjugates in the circulation as sulates.

Both liver and adrenal glands express SULT2A1, an enzyme that has the activity to sulfonate both steroids [START_REF] Richies | Qualitiative evaluation of the expression and activity of five major sulfotransferases (SULTs) in human tissue: the SULT "pie[END_REF]. However, tracer studies indicate these sullates are synthesised by the human adrenal gland rather than the liver [START_REF] Casey | Origin and production rates of deoxycorticosterone and deoxycorticosterone sulfate in men and nonpregnant women[END_REF][START_REF] Labrie | Intracrinology[END_REF]; hepatic SULT2A1 appears to be responsible for the disposal of xenobiotics (Senggunpai, Yoshina and Yamazoe, 2009). The known specificity of SULT2A1 and its expression in H295 cells indicate that this enzyme is responsible for DOC and DHEA sulfation in these cells and is therefore a likely target of GA. The inhibition of steroid sulfation by GA appears to be a direct effect on enzyme activity rather than a decrease in SULT2A1 gene expression. Conjugation of radiolabeled DOC and DHEA was reduced 8h after addition of GA at a concentration which did not affect SULT2A1 mRNA expression.

Others have also reported a disparity between SULT2A1 activity and gene expression and in H295 cells. [START_REF] Kumar | Effect of insulin and testosterone on androgen production and transcription of SULT2A1 in the NCI-H295R adrenocortical cell line[END_REF] showed that testosterone and insulin treatment caused a marked inhibition of DHEA sulfation despite a three fold increase in SULT2A1 expression.

In our experiments we suggest that GA is a competitive inhibitor of SULT2A1 activity and its effects are therefore independent of transcriptional or translational control. The effects of GA in vitro on H295 cells may differ from those expected in the adrenal gland in vivo. SULT2A1 is uniformly expressed by H295 cells so that an increase in free steroid availability caused by inhibition of conjugation will provide substrate for alternative competing enzymes. In contrast adrenal SULT2A1 levels vary at different stages of life and are localised to specific regions of the cortex [START_REF] Rainey | Dissecting human adrenal androgen production[END_REF]. In adulthood, for example, plasma DHEAS levels are highest at age 20-30 years and SULT2A1 expression are localised predominantly in the zona reticularis. Given that mineralocorticoids, glucocorticoids and androgens are produced by different zones of the cortex, it follows that GA effects on salivary steroid levels may reflect zone-specific effects on different steroidogenic enzymes

Conclusions

We have demonstrated that liquorice increases salivary DHEA, DOC and testosterone levels as well as showing expected inhibition of cortisol to cortisone conversion. In vitro analysis of steroidogenesis by H295 cells treated with GA show that in vivo DHEA and DOC effects are probably due to inhibition of steroid conjugation by the adrenal gland. Although these GA effects are not caused by changes in the transcription of SULT2A1 (the enzyme responsible for DOC and DHEA sulfation), it seems clear that the control of SULT2A1 activity, whether genomic or non-genomic, plays an important part in directing pathways of mineralocorticoid, glucocorticoid and androgen biosynthesis.

  Abbreviations

  .

  ]-DHEA (GE Healthcare, Amersham Bucks, UK) and [ 3 H]-DOC (MP Biomedicals,

  A c c e p t e d M a n u s c r i p t 10 significantly correlated only in males (r = 0.4, P = 0.002; females: r = 0.2, P = 0.1). Increases in deoxycorticosterone, DHEA and cortisol could be explained by effects of liquorice on adrenocortical steroidogenesis, particularly as their routes of metabolism are different.

Figure 1 :

 1 Figure Legends

Figure 2 :

 2 Figure 2: Salivary steroid levels throughout the day with non-liquorice confectionery

Figure 3 :

 3 Figure 3: Effects of glycyrrhetinic acid on steroidogenesis in H295 cells incubated with

Figure 4 :

 4 Figure 4: Concentration-dependent effects of glycerrhetinic acid on DOC and DHEA

Figure 5 :

 5 Figure 5: Concentration-dependent effects of glycyrrhetinic acid on 11β-hydroxysteroid

Figure 6 :

 6 Figure 6:.Concentration-dependent inhibition of radiolabeled DOC and DHEA conjugation by glycyrrhetinic acid. H295 cells were incubated for 24h with 3 H DOC or 3 H

Figure 7 :

 7 Figure 7: Temporal effects of glycyrrhetinic acid on DHEA conjugation and SULT2A1

Table 1 . Probability values for effects of liquorice, time of sampling and sex on salivary steroid levels.

 1 Also shown are values for interactions between treatment and sample time and treatment and sex. There were no significant differences for a three way interaction between treatment, sample time and sex.

	22

Table 2 . Effects of liquorice on free and conjugated salivary steroid levels from male volunteers.

 2 Saliva samples (0.5ml) collected at 09.00, 12.00 and 19.00h were pooled and fractionated with Sep-Pak C18 cartridges as described in the methods section. Conjugated steroids were hydrolysed with Helix pomatia prior to assay. Results are means ± SEM.

Acknowledgements

CJK is supported by a British Heart Foundation Centre of Research Excellence award. Funds for the development and validation of assays were provided by an MRC Program Grant awarded to CJK and by the World Anti-doping Agency to EAD. None of the funding bodies had any involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication.

Disclosure statement

The authors have nothing to disclose.

Conflict of interest statement

The authors have declared no conflict of interest.


Waldemar Kanczkowski
email: waldemar.kanczkowski@uniklinikum-dresden.de

Piotr Tymoszuk

Triantafyllos Chavakis

Volker Janitzky

Torsten Weirich

Kai Zacharowski

Monika Ehrhart-Bornstein

Stefan R Bornstein

Kanczkowski Waldemar

Upregulation of TLR2 and TLR4 in the human adrenocortical cells differentially modulates adrenal steroidogenesis. 1

Keywords: Adrenal insufficiency, adrenal dysfunction, steroidogenesis, immune-adrenal crosstalk, pattern recognition receptors

Rapid activation of adrenal steroid release plays a pivotal role in an organism's first line of defense during sepsis. Adrenal gland function is often suppressed in critically ill patients and negatively impacts the overall survival rate. Increasingly, experimental and clinical evidence suggests that Tolllike receptors (TLRs), components of the innate immune system, play a key role in the mediation of systemic responses to invading pathogens during sepsis. In the present study, we aimed to elucidate the effect of TLR2, TLR4 and CD14 upregulation on adrenocortical cell steroidogenesis. We found that TLR4 and CD14 but not TLR2 overexpression in NCI-H295R cells inhibited basal and acute cortisol and aldosterone production. This effect could be partially explained by reduced expression of enzymes involved in the synthesis of latter steroids -CYP11B1 and CYP11B2. Together, these data suggest that TLR upregulation in the steroid producing cells may be involved in the adrenal gland dysfunction during sepsis.

A c c e p t e d M a n u s c r i p t

Introduction

Sepsis and septic shock still remain a major cause of death in intensive care units stations worldwide despite recent improvements in their management and treatment. According to recent estimates, the incidence of severe sepsis in the United States of America accounts for 710.000 patients per year with a mortality rate of 28% [START_REF] Angus | Epidemiology of sepsis: an update[END_REF]. Sepsis is a clinical syndrome resulting from a complex interaction between host and infectious agents; bacterial or viral infections are its primary cause. It is characterized by systemic activation of multiple inflammatory pathways, including cytokine networks and coagulation [START_REF] Van Der | Immunotherapy of sepsis[END_REF] and by activation and dysfunction of innate and adaptive branches of the immune system [START_REF] Russell | Management of sepsis[END_REF]. Recently, an increased frequency of sepsis and septic shock due to infections with Gram positive bacteria has been reported [START_REF] Angus | Epidemiology of sepsis: an update[END_REF]. Increasing evidence suggests that Toll-like receptors (TLRs) play a key role in the mediation of systemic responses to invading pathogens during sepsis. TLRs are components of the innate immune system and are responsible for recognizing a variety of microbial pathogens and the initial induction of immune and inflammatory responses. TLRs sense various conserved pathogen-associated molecules expressed by microorganisms such as lipopolysaccharide (LPS) of Gram negative bacteria (sensed by TLR4), or lipoteichonic acid frequently found on cellular walls of Gram-positive bacteria (detected by TLR2). To date, 13 TLRs have been identified in mammals [START_REF] Leulier | Toll-like receptors--taking an evolutionary approach[END_REF]. Due to their ability to initiate an overt inflammation, TLRs are often associated with destruction of various tissues, e.g. during septic shock; which has initiated the pharmaceutical development of many TLR antagonists [START_REF] Hennessy | Targeting Toll-like receptors: emerging therapeutics?[END_REF]. Increasing experimental and clinical evidence has demonstrated importance of TLR expression on both immune and non-immune cells during sepsis. Significant upregulation of TLRs in various organs e.g. lung, liver, kidney or periphery blood has been reported in different models of sepsis and in critically ill patients [START_REF] El-Achkar | Sepsis induces changes in the expression and distribution of Toll-like receptor 4 in the rat kidney[END_REF][START_REF] Tsujimoto | Neutrophil elastase, MIP-2, and TLR-4 expression during human and experimental sepsis[END_REF][START_REF] Williams | Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality[END_REF].

The hypothalamic-pituitary adrenal axis is a key component of the host response to sepsis and adrenal glands are the main effector part of this system [START_REF] Beishuizen | Endotoxin and the hypothalamo-pituitary-adrenal (HPA) axis[END_REF][START_REF] Gallo-Payet | Hypothalamic-pituitaryadrenal axis and multiple organ dysfunction syndrome in critical illness: A special focus on argininevasopresine and apelin[END_REF]. Indeed, rapid activation of adrenal steroid release plays a pivotal role in an organism's first line of defense during this form of severe illness. The critical role of adrenal derived hormones during stress has been demonstrated using adrenolectomized animals, which demonstrated a higher mortality rate then sham operated controls [START_REF] Mccallum | Endotoxin-induced inhibition of steroid binding by mouse liver cytosol[END_REF]. Multiorgan dysfunction syndrome (MODS) is the main cause of death due to sepsis; however the adrenal gland seldom suffers irreversible anatomical damages due to bilateral necrosis and hemorrhage as reported originally by Waterhouse and Friderichsen (Waterhouse, 1911). It is rather, a transient functional impairment in hormone synthesis which is a more common complication of severe sepsis [START_REF] Prigent | Science review: mechanisms of impaired adrenal function in sepsis and molecular actions of glucocorticoids[END_REF]. Indeed, during sepsis relative adrenal gland insufficiency occurs frequently and is responsible for increased mortality [START_REF] Koo | Adrenal insufficiency during the late stage of polymicrobial sepsis[END_REF][START_REF] Sprung | Hydrocortisone therapy for patients with septic shock[END_REF]. Several factors may be responsible for the incidence of adrenal insufficiency in septic [START_REF] Bornstein | Predisposing factors for adrenal insufficiency[END_REF].

During stress conditions such as sepsis, adrenal hormone production is shifted toward glucocorticoid production as a result of increased ACTH release from pituitary. The generalized suppressive influence of glucocorticoids protects the host from a hyperactive inflammatory response, which may be deleterious to cell and organ functions and ultimately in the outcome of critically ill patients [START_REF] Prigent | Science review: mechanisms of impaired adrenal function in sepsis and molecular actions of glucocorticoids[END_REF]. Furthermore, a substantial drop in aldosterone production during sepsis can be identified, regardless of the rise in renin levels, suggesting that additional factors such as elevated cytokines or nerve regulation substantially modulate adrenal steroidogenesis [START_REF] Prigent | Science review: mechanisms of impaired adrenal function in sepsis and molecular actions of glucocorticoids[END_REF]. Although levels of dehydroepiandrosterone (DHEA) and its sulfated form (DHEAS) decrease in the plasma of septic patients, resulting in the increased cortisol to DHEA ratio, the function of androgens during sepsis is not clear. Indeed, androgen supplementation, as opposed to glucocorticoid replacement does not influence the mortality rate of intensive care unit (ICU) patients [START_REF] Marx | Adrenocortical hormones in survivors and nonsurvivors of severe sepsis: diverse time course of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, and cortisol[END_REF]. We have previously shown expression of several TLRs in mouse and human adrenal glands and in primary cultures of adrenocortical cells (Bornstein et al., 2004a;[START_REF] Kanczkowski | Differential expression and action of Toll-like receptors in human adrenocortical cells[END_REF]. Moreover, we found that TLR2, TLR4 or TLR9 ligands when administered to mice induce a strong systemic and local inflammation of adrenal gland with upregulation of the major proinflammatory cytokines such as IL-1 , IL-6 and TNF- (Bornstein et al. 2004b) Interestingly, in the latter study we have found un upregulation of both TLR2 and TLR4 expression in the adrenal glands from mice that received a single dose of LPS or lipoteichonic acid (Bornstein et al., 2004b). However, we also found that TLR2 and TLR4 deficiency results in a dysregulation of adrenal glucocorticoid production and increased mortality of TLR2 and TLR4 deficient mice. Since the latter observation was paralleled by dysregulation of plasma cytokines and an impairment of adrenocortical cells ultrastructure an important link between intact TLR expression and a proper adrenal function has been postulated [START_REF] Zacharowski | Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome[END_REF].

In the current study, we sought to evaluate the importance of TLR2 and TLR4 upregulation previously seen in mice models using human adrenocortical cells with induced TLRs overexpression.

Understanding of the role which TLR2 and TLR4 play in the adrenocortical cells during sepsis is important because an overt activation of TLRs and related cytokine and oxidative stress may directly interfere with the adrenal cortex hormone synthesis leading to adrenal insufficiency often diagnosed in critically ill patients.

A c c e p t e d M a n u s c r i p t 5

Materials and Methods

Cell culture and stimulation

NCI-H295R cells were grown in DMEM-F12 medium (GibcoBRL/Life Technologies GmbH) supplemented with 1% ITS plus (Gibco-Invitrogen, Carlsbad, CA, USA), 5% Nu-Serum I (Becton Dickinson, Heidelberg, Germany) and either 100 U/mL penicillin G/100 µg/mL streptomycin sulfate (normal cells) or 5 g/ml of blasticidine S (cell clones). An equal number of cells were seeded into 24 well plates and grown until 80% confluence was reached. 24h prior to stimulation cells were grown in medium without any supplements and antibiotics. For stimulation cells were incubated in supplementfree medium together with angiotensin II (AII) (10 -7 M) or forskolin (FSK), (10 -6 M) for 24h.

Western blot

Cell proteins were extracted using Cell Lysis Buffer (Cell Signalling Technology, Denvers, MA, USA) according to the manufacturer's instructions. Protein concentration was measured with Bio-Rad Protein Assay Reagent (Bio-Rad, München, Germany) and separated in polyacrylamide gels and electro-transferred onto PVDF membranes. After blotting polyclonal anti-sera against human TLR2 (concentration 1g/ml, ProSci. Inc.); monoclonal antibody against CD14 (clone 5A3B11B5) and polyclonal antibody recognizing TLR4 (H80) (both form Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at 1:250. Signal was developed using Supersignal West Femto Substrate (Thermo-Pierce) and visualized using LAS 3000 Bioimager (Fuji, Tokyo, Japan).

Stable transfection of the NCI-H295R cells with TLR2 and TLR4-CD14 encoding plasmids

NCI-H295R cells were transfected with expression plasmids encoding for TLR2, TLR4-CD14 or with an empty plasmid as a negative control (MOCK) (InvivoGen, San Diego, CA, USA) using Lipofectamine™ LTX PLUS transfection reagent (Invitrogen Life Technologies, Karlsruhe, Germany). NCI-H295R cell clones stably expressing TLR2, TLR4-CD14 or containing an empty plasmid were selected using blasticidin S (5 µg/ml; InvivoGen).

RNA isolation and cDNA synthesis

RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For reverse transcription reaction (RT), 1 μg of mRNA was incubated at 37 °C for 1 h in buffer containing 200 U of M-MLV reverse polymerase, oligo(dT) 15 primers mix and dNTPs. All reagents were from Promega (Madison, USA).

Quantitative PCR

A c c e p t e d M a n u s c r i p t 6 mRNA levels of human cholesterol side-chain cleavage enzyme (CYP11A1) and steroid acute regulatory protein (StAR) genes were determined using the Quantitec SYBR® PCR Master Mix (Qiagen, Hilden, Germany) and Light Cycler® 2.0 PCR thermocycler (Roche Applied Science, Basel, Switzerland). The amplification protocol, primer sequences and annealing temperatures were used as reported previously [START_REF] Kanczkowski | Differential expression and action of Toll-like receptors in human adrenocortical cells[END_REF]. Total cDNA copy numbers of CYP11A1 and StAR genes were calculated as a ratio of copy numbers of amplified gene of interest to copies of internal standard and adjusted to the copies of -actin. Quantitative standard curves were generated applying dilutions over seven log scales (10 1 -10 8 ) per capillary of the pCRII-TOPO vector (Invitrogen Life Science) containing PCR-fragment of the gene of interest and determined by the Light Cycler® quantification software version 3.5.3 (Roche Applied Science). Due to the high homology rate between 11-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, a relative changes in number of both transcripts were evaluated using Taqman PCR Master Mix (Roche) and transcript specific FAM-TAMRA labeled probe. Samples were measured in duplicate and expression was calculated relative to the housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1) using ΔCT method. Primers and probe sequences used were already reported [START_REF] Chai | Steroidogenesis vs. steroid uptake in the heart: do corticosteroids mediate effects via cardiac mineralocorticoid receptors?[END_REF].

Steroid hormone measurement

H295R cells were treated with 10 -7 M AII or 10 -6 M FSK in DMEM-F12 medium. Concentration of secreted adrenal steroids in cell supernatants were measured using specific radioimmunoassays: DSL for aldosterone and DHEA (DSL, Sinsheim, Germany) and DPC for cortisol (DPC Biermann/ Siemens Healthcare Diag., Eschborn, Germany).

Results

Generation of NCI-H295R cell clones with stable enhanced expression of TLR2 or TLR4 proteins.

To elucidate the effect of TLR2 and TLR4 upregulation on adrenocortical cell steroidogenesis and inflammation, we generated several cell clones stably expressing the corresponding proteins as well as containing an empty plasmid (MOCK). Western blot analysis confirmed upregulation of TLR2 and TLR4 expression. TLR2 was found to be successfully upregulated in 100% ( 14 1B). Successful generation of stably transfected NCI-H295R cell clones with an upregulation of TLR4 (1.52-fold) and CD14 by 13.56-fold was recently reported [START_REF] Kanczkowski | Abrogation of TLR4 and CD14 expression and signaling in human adrenocortical tumors[END_REF].

Differential effect of TLR2 and TLR4 overexpression on human adrenocortical cell steroidogenesis

To determine whether an increased expression of TLR observed in septic adrenal glands modulates adrenal steroidogenesis, levels of aldosterone, cortisol and DHEA were measured in supernatants of the NCI-H295R cell clones with stable upregulation of TLR2 and TLR4-CD14 proteins before and after incubation with 10 -7 M of AII or 10 -6 M of FSK. Our results showed that enhanced TLR2 expression mediated a significant 2.5-fold increase in basal aldosterone secretion as compared to MOCK control (89.89 ± 8.02 vs. 35.29 ± 3.27 pg/mL; p < 0.01) (Fig. 2A). Although the potential of AII to enhance aldosterone production did not differ substantially between MOCK and NCI-H295R cells with TLR2 upregulation (18.7-fold vs. 17.7-fold), the resulting concentrations of this mineralocorticoid after incubation with AII varied significantly and was almost 2.7-fold higher in TLR2 than in MOCK group (1680 ± 286 pg/mL vs. 624.4 ± 196.6 pg/mL, p < 0.05) (Fig. 2B).

Interestingly, contrary to TLR2 an enhanced expression of TLR4-CD14 protein reduced basal (5 ± 2.23 vs. 35.29 ± 3.27 pg/mL, 7-fold, p < 0.05) and AII-mediated aldosterone production (24.34 ± 8.4 vs. 624.4 ± 196.6 pg/mL, 4.8-fold, p < 0.05) Fig. 2A. In contrast to aldosterone, basal levels of cortisol varied slightly but not significantly (1.18-fold increase) between NCI-H295R cells with an enhanced TLR2 expression and MOCK control cells (89.86 ± 8.48 vs. 75.97 ± 9.78 nmol/L) (Fig. 2C). No significant difference was observed after FSK treatment between the latter mentioned 2 cell groups.

Basal (6.7 ± 1.57 vs. 75.97 ± 9.78 nmol/L, p < 0.01) and FSK-mediated cortisol production (15.0 ± 3.09 vs. 406 ± 108 nmol/L, 27-fold, p < 0.01) were strongly reduced in NCI-H295R cells with enhanced TLR4-CD14 expression as compared to the MOCK control cells (Fig. 2D). Basal DHEA levels increased in NCI-H295R cells after enhanced expression of both TLR2 (8.94 ± 2.2 vs. 5.46 ± 1.64 ng/mL, p < 0.01) and TLR4-CD14 proteins (11.61 ± 1.65 vs. 5.46 ± 1.64 ng/mL) (Fig. 2E).

However, despite this initial increase in FSK-mediated DHEA production was lower in both TLR2 (24.49 ± 6.83 vs. 42.9 ± 14.26 ng/mL) and in TLR4-CD14 (33.83 ± 3.54 vs. 42.9 ± 14.26 ng/mL) as compared to MOCK transfected cells (Fig. 2F).

Expression of steroidogenic enzymes and proteins involved in cholesterol utilization

To evaluate the effect of the enhanced expression of TLR2 and TLR4 on adrenal steroidogenesis, expression of key enzymes involved in cholesterol transport (StAR) as well as steroid biosynthesis (CYP11B1, CYP11B2 and CYP11A1) were assessed (Fig. 3). Our results show that in comparison to A c c e p t e d M a n u s c r i p t control cells, stimulation with AII induced CYP11B2 gene expression in NCI-H295R cells with enhanced TLR2 expression (62.04 ± 8.265 vs. 50.99 ± 6.29; 1.2-fold), whereas in cells with enhanced TLR4-CD14 expression it caused an opposite effect (21.55 ± 2.316 vs. 50.99 ± 6.29; 2.36-fold, p < 0.05) (Fig. 3A). Enhanced upregulation of both TLR2 (8.359 ± 2.55 vs. 14.57 ± 3.72,p < 0.05) reduced FSK-induced CYP11B1 expression as compared to the MOCK control Fig. 3B. Analysis of CYP11A1 expression showed that NCI-H295R cells with upregulation of TLR2 tend to have higher copy numbers of this gene (0.41 ± 0.1 vs. 0.22 ± 0.026) as compared to MOCK and to cells with upregulation of TLR4-CD14 (0.41 ± 0.1 vs. 0.08 ± 0.008, p< 0.05). In fact, basal expression of CYP11A1 gene was significantly reduced in NCI-H295R cells after TLR4-CD14 upregulation compared with TLR2 upregulation (0.41 ± 0.1 vs. 0.08 ± 0.008, p < 0.05). Finally, an upregulation of TLR2 in NCI-H295R cells resulted in higher StAR copy number as compared to the MOCK control, however the differences were not significant (0.263 ± 0.0137 vs. 0.19 ± 0.002, 1.38-fold) Fig. 3D. On the contrary, enhanced expression of TLR-CD14 resulted in reduced StAR mRNA levels (0.14± 0.016 vs. 0.19 ± 0.002, 1.36-fold) Fig. 3D. Basal levels of StAR mRNA were significantly higher in TLR2 (1.8-fold, p < 0.05) than in TLR4-CD14 clones of NCI-H295R cells.

Discussion

Severe sepsis seldom causes irreversible anatomical damages to the adrenal gland leading to primary adrenal failure. Instead, a transient impairment in the hormone synthesis is a commonly found complication [START_REF] Prigent | Science review: mechanisms of impaired adrenal function in sepsis and molecular actions of glucocorticoids[END_REF]. Compelling evidence strongly implicates expression of various TLRs and TLR-signaling in many cell types with the progression of sepsis and septic shock [START_REF] Salomao | Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock[END_REF]. Being the most important group of the innate immunity receptors, TLRs are primarily responsible for pathogen recognition and initiation of an overt inflammatory responses which often culminate in systemic inflammatory response syndrome and septic shock. Although sepsis may be caused by various microorganisms, both Gram positive and Gram negative bacteria, recognized by either TLR2 or TLR4, are the most frequently isolated pathogens from the blood of septic patients.

TLR overexpression in certain tissues during course of sepsis is not unexpected. Indeed, during sepsis and septic shock an enhanced TLR expression was already documented e.g. TLR2 and TLR4 in liver [START_REF] Tsujimoto | Neutrophil elastase, MIP-2, and TLR-4 expression during human and experimental sepsis[END_REF] or TLR2, TLR4 and TLR9 in peripheral blood [START_REF] Brandl | TLR4 surface display on human monocytes is increased in septic patients[END_REF]. This finding is in line with our previous results showing an upregulation of TLR2 and TLR4 in the adrenal glands of mice after injection of lipotechonic acid and LPS (Bornstein et al., 2004b;[START_REF] Zacharowski | Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome[END_REF]. In order to see whether an increased expression of TLRs in adrenocortical cells may impact adrenal steroidogenesis, we have stably transfected NCI-H295R cell with expression plasmid encoding either for TLR2 or TLR4-CD14 and tested basal and AII or FSK-stimulated hormone synthesis in these transfected cells. Upregulation of TLR2 results in enhanced basal and AII-mediated aldosterone A c c e p t e d M a n u s c r i p t 9 synthesis, having no effect on cortisol or DHEA levels. Interestingly, upregulation of TLR4 in NCI-H295R cells strongly reduced aldosterone and cortisol synthesis at basal conditions and after stimulation with either FSK or AII. However, at the same time it showed no effect on DHEA production. These results suggest that overexpression of TLRs may differentially modulate adrenocortical steroidogenesis in a direct manner which is very interesting especially considering our in vivo data on TLR-upregulation in mouse adrenal glands during SIRS. Moreover, it shows that not only TLR4-deficiency but also TLR4 enhanced expression affects adrenal glucocorticoid production. This seemingly contradictory observation could be explained by differences between in vivo and in vitro conditions. TLR4-deficient mice have an increased basal cortisol production due to most probably elevated levels of proinflammatory cytokines and marked hyperplasia of the adrenal cortex [START_REF] Zacharowski | Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome[END_REF]. We have tested NCI-H295R cells, since these cells are the only human adrenocortical cell line which expresses all enzymes necessary to synthesize three major adrenal steroids aldosterone, cortisol and DHEA [START_REF] Rainey | Adrenocortical cell lines[END_REF]. However, using a malignant transformed cell line has some disadvantages such as insensitivity to adrenocorticotropic hormone action or reduced expression of certain TLRs [START_REF] Kanczkowski | Differential expression and action of Toll-like receptors in human adrenocortical cells[END_REF]. In fact we found reduced expression of TLR2 and abrogation of TLR4-CD14 in NCI-H295R cells compared to primary cell cultures [START_REF] Kanczkowski | Abrogation of TLR4 and CD14 expression and signaling in human adrenocortical tumors[END_REF]. Since antibiotic selection or transfection conditions had minimal effect on the steroidogenesis of NCI-H295R cells transfected with TLR2 or an empty control plasmid; it is therefore unlikely that these conditions could be responsible for the observed TLR4-CD14mediated aldosterone and cortisol reduction. Thus, one of the possible mechanisms involved in this effect may be reduced expression of enzymes involved in biosynthesis of cortisol (CYP11B1) and aldosterone (CYP11B2). Indeed, our results show that compared to MOCK or TLR2 transfected cells, NCI-H295R cells with enhanced expression of TLR4-CD14 had reduced capacity to induce the expression of both enzymes. Finally, reduced steroidogenesis may also result from impairment in transport and metabolism of the main steroid substrate cholesterol. To test this possibility, we analyzed the expression of 2 main enzymes involved in cholesterol utilization: StAR and CYP11A1. Although the mRNA levels of the latter enzymes did not differ between MOCK and TLR4CD14 transfected NCI-H295R cells, it was significantly reduced when compared to cells transfected with TLR2. This observation suggests that at least in part, the differences in aldosterone and cortisol synthesis between TLR2 and TLR4 upregulation may arise from the different expression level of the mentioned cholesterol metabolizing enzymes. We did not, however, find any correlation between the upregulation of TLR2 and StAR expression on protein level (data not shown). It has already been reported that LPSmediated activation of TLR4 in primary rodent adrenocortical cells reduces basal and ACTH-mediated corticosterone synthesis; this effect was attributed to an increased level of reactive oxygen species [START_REF] Garcia | Calcium and reactive oxygen species as messengers in endotoxin action on adrenocortical cells[END_REF]. Since the primary rodent adrenocortical cells may contain some amount of the immune cells, the involvement of the latter cells in reactive oxigen species-mediated steroidogenesis dysfunction could not be excluded. Therefore, our data using NCI-H295R cell line which is a pure In conclusion, our results demonstrate that increased expression of TLR4 but not TLR2 in NCI-H295R cells can negatively impact adrenocortical cell hormone synthesis and expression of enzymes involved in steroidogenesis and cholesterol metabolism. In addition these data suggest that an overt activation of the TLR system in the steroid producing cells be potentially involved in the development of the critical illness related corticosteroid insufficiency in the long-term course of sepsis.

Acknowledgements

We are grateful for Prof. Manfred Wirth (Department of Urology, Technical University Dresden, Dresden) for generously providing tissue samples. We greatly appreciate the help from Uta Buro for performing real-time PCR. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to Stefan R. Bornstein (BO 1141/8-4) and to Kai Zacharowski (ZA 243/11-4).

Legends

A c c e p t e d M a n u s c r i p t

  a suppression of the HPA axis by exogenous glucocorticoids, viral infections or certain medication

  /14) of randomly analyzed clones, TLR4-CD14 in 42% (3/7). For further experiments 3 clones with the highest expression of TLRs representing each group were used. A Western blot analysis of TLR2 expression obtained from a representative cell clone is depicted in Figure 1A. Densitometrical quantification of TLR2 expression performed on 2 representative NCI-H295R cell clones showed a 3.75-fold (clone A c c e p t e d M a n u s c r i p t 7 #89) and a 3.25-fold (clone #102) upregulation of protein expression as compared to the MOCK transfected clone (Figure

  cells could partially support the notion that LPS-mediated TLR4 upregulation in steroid producing cells may negatively impact adrenal steroidogenesis. Whether reactive oxygen species are involved in an observed by us TLR4-mediated downregulation of adrenal steroidogenesis is the subject of undergoing studies in our laboratory.

Figure 1 .

 1 Figure 1. Generation of NCI-H295R clones with stable upregulation of TLR2 and TLR4-CD14. (A) Western blot examination of TLR2 expression in TLR2-and MOCK-transfected NCI-H295R cells. TLR2 expression is strongly upregulated in both representative cell clones as compared with MOCKcontrol cells. (B) Densitometrerical analysis of Western blot results showing 3.75-fold (#89) and 3.25-fold (#102) upregulation of TLR2 protein expression in 2 representative NCI-H295R cell clones. Expression of TLR2 was adjusted to expression of tubulin.

Figure 2 .

 2 Figure 2. The effect of TLR2 and TLR4-CD14 upregulation on adrenal gland steroidogenesis. Stable upregulation of TLR2 in NCI-H295R cells resulted in an increased (A) basal and (B) angiotensin (AII)-induced aldosterone production. Conversely, TLR4-CD14 upregulation resulted in a decreased (A) basal and (B) FSK-induced aldosterone production and strongly reduced (C) basal and (D) FSK-mediated cortisol production. Neither TLR2 nor TLR4 upregulation did modulate adrenal (E) basal or (F) FSK-mediated production of DHEA as compared to MOCK control cells. Absolute values of hormone levels were evaluated in 3 different clones of NCI-H295R cells with the highest upregulation of either TLR2 or TLR4-CD14. Pooled values from three independent experiments are depicted as mean ± S.E.M. (*) p<0.05, (**) p<0.01.

Figure 3 .

 3 Figure 3. Effect of TLR2 and TLR4-CD14 upregulation on the expression of steroidogenic enzymes and proteins involved in cholesterol utilization. Stable upregulation of TLR4-CD14 but not TLR2 in NCI-H295R cells resulted in decreased (A) angiotensin II (AII) (B) and FSK-induced CYP11B2 and CYP11B1 gene expression. Relative values of CYP11B2 and CYP11B2 expression have been adjusted to the expression of house-keeping gene HPRT-1. Effect of TLR2 and TLR4 upregulation on the absolute mRNA levels of (C) CYP11A1 and (D) StAR genes normalized to expression of beta actin gene. Values from three independent experiments depicted as mean ± S.E.M. (*) p<0.05, (**) p<0.01.

  Figure 1 Click here to download high resolution image

  Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image


Alex Lindsay-Scott

Ian G Wood
email: ian.wood@ucl.ac.uk

David P Dobson

Lidunka Vočadlo

John P Brodholt

Wilson Crichton

Michael Hanfland

Takashi Taniguchi

The Isothermal Equation of State of CaPtO 3 Post-perovskite to 40 GPa

ABX 3 post-perovskite phases that are stable (or strongly metastable) at room-pressure are of importance as analogues of post-perovskite MgSiO 3 , a deep-Earth phase stable only at very high pressure. Commonly, CaIrO 3 has been used for this purpose, but it has been suggested that CaPtO 3 might provide a better analogue. We have measured the isothermal incompressibility, at ambient temperature, of orthorhombic post-perovskite structured CaPtO 3 to 40 GPa by X-ray powder diffraction using synchrotron radiation. A third-order

Birch-Murnaghan equation of state fitted to the experimental data yields V 0 = 228.10(2) Å 3 , K 0 = 168.2(8) GPa and K' 0 = 4.51 (6). Similar fits to the cube of each axis of the unit cell shows that the b-axis is the most compressible (b 0 = 9.9191(5) Å, K 0 = 123.3(5) GPa, K' 0 = 2.37(3)); the a-axis (a 0 = 3.12777(8) Å, K 0 = 195.7(8) GPa, K' 0 = 6.63( 8)) and c-axis (c 0 = 7.3551(4) Å, K 0 = 192(2) GPa, K' 0 = 12.2(3)) are both much stiffer and have almost identical incompressibilities when the material is close to ambient pressure, but the c-axis shows greater stiffening on compression. Comparison of these axial incompressibilities with those of CaIrO 3 shows that CaPtO 3 is slightly less anisotropic under compression (possibly A c c e p t e d M a n u s c r i p t because of the absence of Jahn-Teller distortion), suggesting that CaPtO 3 may be a somewhat better analogue of MgSiO 3 .

Our sample also contained minor amounts of a cubic Ca x Pt 3 O 4 phase, for which the third-order Birch-Murnaghan equation of state parameters were found to be: V 0 = 186.00(3) Å 3 , K 0 = 213(1) GPa and K' 0 = 4.9(1).

Keywords:

CaPtO 3 CaPt 3 O 4
Post-perovskite Equation of state X-ray powder diffraction A c c e p t e d M a n u s c r i p t

Introduction

Perovskite-structured (PV) MgSiO 3 transforms to an orthorhombic CaIrO 3 -structured postperovskite (PPV) phase at around 120 GPa [START_REF] Murakami | Post Perovskite Phase Transition in MgSiO 3[END_REF][START_REF] Oganov | Theoretical and Experimental Evidence for a Post-Perovskite Phase of MgSiO 3 in Earth's D'' layer[END_REF].

The atomic arrangement in the PPV-phase of MgSiO 3 differs greatly from that found in PV-MgSiO 3 and thus one might expect that the physical properties of the two phases will also differ appreciably. The perovskite phase of MgSiO 3 contains the 3-dimensional network of corner-linked octahedra found in all perovskites. In contrast, the structure of ABO 3 postperovskites is commonly described in terms of sheets of corner-and edge-sharing octahedra, lying parallel to (010), separated by planar A-cation interlayers. These features are illustrated in Figure 1, which shows the crystal structure of the post-perovskite phase of CaIrO 3 , viewed down the a-axis; however, as discussed further below (Section 4) this view of the postperovskite structure in terms of layers is almost certainly too simplistic.

The pressure at which the phase transition in MgSiO 3 occurs implies that post-perovskite MgSiO 3 might be the majority phase in the D'' region that extends into the mantle from the core-mantle boundary. If this is the case, then the physical and chemical properties of postperovskite are likely to dominate the dynamics of much of the core-mantle boundary region.

Measurements of the physical and chemical properties of post-perovskite MgSiO 3 are difficult to perform because it is stable only at megabar pressures and so it has been more practical to obtain some of the experimental results required for comparison with computer simulations of PPV-structured MgSiO 3 from isostructural analogues whose compressive and thermal distortion at lower temperature and pressure are similar to those of MgSiO 3 .

Analogues studied to date include CaIrO 3 [START_REF] Boffa Ballaran | Equations of state of CaIrO 3 perovskite and post-perovskite phases[END_REF][START_REF] Lindsay-Scott | Thermal expansion of CaIrO 3 determined by X-ray powder diffraction[END_REF]Martin et al., 2007Martin et al., , 2007a;;[START_REF] Martin | The local post-perovskite structure and its temperature dependence: atom-pair distances in CaIrO 3 revealed through analysis of the total X-ray scattering at high temperatures[END_REF][START_REF] Sugahara | Single-crystal X-ray diffraction study of CaIrO 3[END_REF][START_REF] Hirai | The crystal structure of CaIrO 3 post-perovskite revisited[END_REF] and CaPtO 3 [START_REF] Inaguma | High-pressure synthesis, structure, and characterization of a post-perovskite CaPtO 3 with CaIrO 3 -type structure[END_REF][START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF]. CaIrO 3 may be synthesised at atmospheric pressure (see e.g. [START_REF] Lindsay-Scott | Thermal expansion of CaIrO 3 determined by X-ray powder diffraction[END_REF]. To date, synthesis of CaPtO 3 has A c c e p t e d M a n u s c r i p t been reported only at high pressures, in the range 4 GPa [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF] to 7 GPa [START_REF] Inaguma | High-pressure synthesis, structure, and characterization of a post-perovskite CaPtO 3 with CaIrO 3 -type structure[END_REF]. However, after synthesis, CaPtO 3 is readily recoverable to atmospheric pressure, where it remains stable, or at least very strongly metastable; for example, high-temperature X-ray powder diffraction measurements in our laboratory (unpublished) have shown that it persists, in air, for periods of longer than one hour at temperatures as high as 800 o C. CaPtO 3 is of interest because, unlike CaIrO 3 , it does not exhibit structural distortion due to the Jahn-Teller effect and thus it has been suggested that it might provide a better analogue of MgSiO 3 [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF]. Boffa [START_REF] Boffa Ballaran | Equations of state of CaIrO 3 perovskite and post-perovskite phases[END_REF] and Martin et al. (2007) have measured the bulk and axial incompressibilities of CaIrO 3 , but CaPtO 3 has been studied only at ambient pressure and temperature [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF][START_REF] Inaguma | High-pressure synthesis, structure, and characterization of a post-perovskite CaPtO 3 with CaIrO 3 -type structure[END_REF]. In this paper we report measurements of CaPtO 3 to 40 GPa in order to assess the similarity of its behaviour under compression to that of CaIrO 3 and MgSiO 3 .

Experimental method

The sample was synthesized under high pressure and high temperature using a belt-type high-pressure apparatus at the National Institute for Materials Science, Tsukuba, Ibaraki, Japan. The starting material was a stoichiometric powder mixture of PtO 2 and CaO, ground together in a mortar and sealed in a Pt capsule. This was then compressed to 4 GPa, heated to 800 o C for 24 hours and then quenched to room temperature prior to slow decompression.

The recovered dark-yellow powder was characterized by X-ray powder diffraction. The sample for this X-ray analysis was prepared as a thin smear on an "off-axis" silicon plate (i.e. one cut so as to produce no Bragg reflections) and the powder pattern was recorded using a et al., 1989). The lattice parameters for the CaPtO 3 (estimated from a LeBail refinement of the data, see below) were found to be in reasonable agreement with those previously reported at ambient pressure and temperature (see Table 1a). Our values are much closer to those of [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF] than those of [START_REF] Inaguma | High-pressure synthesis, structure, and characterization of a post-perovskite CaPtO 3 with CaIrO 3 -type structure[END_REF]; however, when comparing these results it should be remembered that, due to the lack of an internal standard and limited 2θ range, they are likely to be less accurate on an absolute scale than their precision might otherwise suggest (although it should, in principle, have been possible to use the platinum present in our sample as an internal standard, in practice there was insufficient platinum present to allow this to be done reliably in any of our experiments).

A selected portion of the sample was loaded, with a helium pressure medium, into a 150 μm diameter hole in a 40 μm thick steel gasket mounted between two opposed 300 μm diamond culets of a Boehler-Almax-type membrane-driven diamond anvil cell. Pressures were determined from the shift of the wavelength of the ruby-R 1 fluorescence line, using the recent pressure-scale calibration of [START_REF] Jacobsen | Compression of single-crystal magnesium oxide to 118 GPa and a ruby pressure gauge for helium pressure media[END_REF] for helium pressure media. Ruby fluorescence measurements were made before and after collection of each diffraction pattern, with the ruby-R 1 wavelength at the data point taken as the mean value of these two readings.

The uncertainty in the pressure, due to relaxation of the cell during the collection of each diffraction pattern, was estimated from the difference in the two ruby measurements, in combination with the quoted uncertainty in the B-parameter in the equation relating the pressure to the ruby-R 1 wavelength-shift [START_REF] Jacobsen | Compression of single-crystal magnesium oxide to 118 GPa and a ruby pressure gauge for helium pressure media[END_REF]; see Table 1 for further details.

A c c e p t e d M a n u s c r i p t

Diffraction patterns were collected on the ID09 beamline at the ESRF, using a monochromatic wavelength of 0.41456 Å on a mar555 image plate detector. The detector was positioned at 358.71 mm from the sample, so as to cover a range 0 o < 2θ < 37.9 o ; the exposure time was ~ 1 second. Data were collected with increasing pressure from 2.90(2) GPa to 42.50(4) GPa, with a final pattern recorded at 1.21(1) GPa after decompression. The 2θ vs Intensity patterns used in the powder refinement were obtained by integrating around the Debye-Scherrer rings, after correction for detector distortion and tilt, using the Fit2D package [START_REF] Hammersley | Calibration and correction of distortion in two dimensional detector systems[END_REF]. The diffraction patterns were then fitted, over the range 3.16 o < 2θ < 33.2 o , to obtain unit-cell parameters using the Le Bail method (Le Bail et al., 1988) implemented in the GSAS suite of programs [START_REF] Larson | General Structure Analysis System (GSAS)[END_REF] with the EXPGUI graphical interface [START_REF] Toby | EXPGUI, a graphical user interface for GSAS[END_REF]. The lineshape used in the GSAS refinements was "profile function number 2", which is based on a pseudo-Voigt function [START_REF] Larson | General Structure Analysis System (GSAS)[END_REF]. At pressures above 12.6 GPa diffraction peaks characteristic of hexagonal (P6 3 /mmc) He crystals were observed [START_REF] Mao | High-pressure phase diagram and equation of state of solid helium from single-crystal X-ray diffraction to 23.3 GPa[END_REF] and so this phase was also included in the refinements. Examples of the fitted diffraction patterns at 2.90(2) GPa and 42.50(4) GPa are shown in Figure 2. It can be seen that the Bragg reflections in Fig. 2B are broader, due to the development of non-hydrostatic stresses; after decompressing the sample the widths of the reflections returned to their original low-pressure values. The onset of this line broadening is clearly indicated in the refined values of the GSAS profile parameters; these are effectively invariant until ~16 GPa, but at higher pressures the values of some of the profile coefficients alter systematically. In particular, the coefficient U (which affects the Gaussian variance of the Bragg peaks via a term in Utan 2 (θ); [START_REF] Caglioti | Choice of collimators for a crystal spectrometer for neutron diffraction[END_REF] increases, with the increase becoming marked above ~22 GPa, an effect that can be attributed to nonuniform strain in the crystallites [START_REF] Larson | General Structure Analysis System (GSAS)[END_REF]. Possible sources of nonhydrostatic stresses in the experiment are solidification of the He pressure medium and grain-grain interactions in the CaPtO 3 sample. This line broadening does not appear to lead A c c e p t e d M a n u s c r i p t to any systematic errors in the refined values of the cell parameters (see Figs 3 and4), but it does increase their estimated uncertainties, which are greater by about a factor of seven at ~40 GPa than at pressures below ~7 GPa (Table 1b).

Results

The observed unit-cell parameters and unit-cell volume of CaPtO 3 between 1.21(1) GPa and 42.50(4) GPa are listed in Table 1b. Figure 3 shows the change in volume with pressure and Figure 4 shows the relative compression of the three unit-cell edges, plotted as the cube of the axial compression ratios. It can be seen from Figure 4 that the compression is strongly anisotropic, being greatest for the b-axis (i.e. for the direction perpendicular to the sheets of PtO 6 octahedra and Ca-ion interlayers) and smallest for the c-axis (i.e. along the chains of apex-linked PtO 6 octahedra). The data, including the decompression data point, both for the unit-cell volume and for the cubes of the unit-cell axes, were fitted to third-order Birch-Murnaghan equations of state [START_REF] Birch | Finite strain isotherm and velocities for single crystal NaCl at high pressures and 300 degrees K[END_REF] by non-linear least-squares using the EOS-fit program [START_REF] Angel | Equations of State[END_REF](Angel, , 2001)); the weighting scheme in the refinement used weights calculated from the errors in both the pressure and in the unit-cell volume and lattice parameters. The resulting values of the three equation-of-state parameters for each of the four fits are listed in Table 2. The values of V 0 , a 0 , b 0 , and c 0 thereby obtained are consistent both with the lattice parameters of CaPtO 3 from previous powder diffraction studies at ambient pressure [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF][START_REF] Inaguma | High-pressure synthesis, structure, and characterization of a post-perovskite CaPtO 3 with CaIrO 3 -type structure[END_REF] and with those that we obtained in the present study during the sample characterisation (there is, however, clearly a slight systematic offset between our two diffraction experiments as all of the values from the high-pressure study are slightly higher, by 0.1% for the unit-cell volume and 0.02 -0.07 % for the unit-cell edges). The good internal consistency of the high-pressure data sets can be demonstrated in two ways. Firstly, the values from the non-linear least-squares fit of the volumetric incompressibility (K 0 ) and of its first derivative with respect to pressure (K' 0 ) of (see e.g. [START_REF] Angel | Equations of State[END_REF] that is shown as an inset in Figure 3. In this method of analysis, the incompressibility at zero pressure is obtained from a weighted linear fit of the normalised

stress 2 5 2 1 3 ) f ( f P F E E E   against finite strain,                 1 5 0 3 2 0 V V . f E
. Secondly, the value of V 0 obtained from the product of a 0 , b 0 , and c 0 is 228.19(2) Å 3 , which differs by 0.09(3) Å 3 from that obtained when the volume is fitted directly.

In addition to the CaPtO 3 , there was sufficient of the cubic Ca x Pt 3 O 4 phase present in the sample to allow reliable determination of its cell parameter. By fitting the unit-cell volume to a third-order Birch-Murnaghan equation of state, as described above, we obtained the following values: V 0 = 186.00(3) Å 3 ; K 0 = 213(1) GPa; K' 0 = 4.9(1). When considering these results, which are reported here for completeness, it should, however, be remembered that this material has been reported to be of variable stoichiometry, with 0 ≤ x ≤ 1 [START_REF] Bergner | Neue Verbindugen vom Na x Pt 3 O 4 -Strukturtyp[END_REF].

Discussion

The Although LDA calculations commonly overestimate the binding of the atoms and hence lead to calculated unit-cell parameters that are smaller than the experimental values, the discrepancy of 7% in V 0 is very large; similarly the value of K 0 from the computer simulations is over 30% greater than our experimental value (note, however, that if the equation-of-state parameters of Matar et al. are used to calculate K at the experimental value of V 0 a value of 157 GPa is obtained, in much closer agreement with our experimental result).

The reasons for this poor agreement are not clear, especially as [START_REF] Matar | Ab initio investigation of perovskite and post-perovskite CaPtO 3[END_REF] also state that test calculations carried out using the generalised gradient approximation (GGA), which commonly leads to an overestimate of the unit-cell volume, did not produce any significant improvement in the agreement between their calculated volume and the available experimental results. It is, perhaps, possible that the discrepancy is simply due to the lack of state points in the calculations, which appear to have been carried out at only six volumes, of which only two were for the material under compression. Unfortunately, no lattice parameters are given by [START_REF] Matar | Ab initio investigation of perovskite and post-perovskite CaPtO 3[END_REF], only unit-cell volumes, and so it is impossible to determine whether their calculations produced a general underestimate of all three cell parameters or whether they failed to reproduce correctly the axial ratios of the crystal.

It is also of interest to compare the present results with the experimental values of the equation-of-state parameters for the other low-pressure post-perovskite analogue phase, CaIrO 3 . We have found that PPV-CaPtO 3 has a slightly larger unit-cell volume and lower value of K 0 than PPV-CaIrO 3 . Boffa Ballaran et al. ( 2007) fitted a third-order Birch-Murnaghan equation to single-crystal X-ray data from PPV-CaIrO 3 , obtaining V 0 = 226.38(1) Å 3 and K 0 = 181(3) GPa, with K' 0 = 2.3(8). Although the uncertainty is large, the value of K' 0 for CaIrO 3 is significantly lower than our corresponding value for CaPtO 3 . Boffa Ballaran et al. ( 2007) used methanol-ethanol as their pressure medium, and the ruby scale of [START_REF] Mao | Calibration of the ruby pressure gauge to 800 kbar under quasi-hydrostatic conditions[END_REF]; however, the difference in K' 0 cannot be attributed simply to differences A c c e p t e d M a n u s c r i p t in the pressure scale, since if we apply the ruby scale of [START_REF] Mao | Calibration of the ruby pressure gauge to 800 kbar under quasi-hydrostatic conditions[END_REF] to our data (see Table 2) we still obtain a value of K' 0 that is significantly higher than that of CaIrO 3 . Other previous measurements of the compression of PPV-CaIrO 3 are those obtained using X-ray powder diffraction by Martin et al. (2007) who fitted a second-order Birch-Murnaghan equation, giving V 0 = 226.632(45) Å 3 and K 0 = 180.2(3) GPa; our corresponding values for PPV-CaPtO 3 (V 0 = 227.95(2) Å 3 , K 0 = 174.0(5) GPa, with K' 0 fixed at 4; see Table 2) show an increase in V 0 and decrease in K 0 similar to that discussed above, although, as K' 0 is fixed at 4 in both cases, the differences in the two fitted parameters are now not as large.

A further contrast in the behaviour of CaPtO 3 and CaIrO 3 lies in the relative unit-cell volumes and incompressibilities of the PPV-and PV-structured forms of the two materials.

For CaIrO 3 , Boffa Ballaran et al. ( 2007) found experimentally that, at zero pressure, the ratio of the volumes of the PPV-and PV-structured phases was 0.98656( 6) and that the PVstructured phase, with K 0 = 198(3) GPa and K' 0 = 1.2(8), was stiffer (at ambient pressure) than the PPV-structured phase. For CaPtO 3 , the computer simulations of [START_REF] Matar | Ab initio investigation of perovskite and post-perovskite CaPtO 3[END_REF],

showed that, at zero pressure, the PPV-and PV-structured phases have a similar volume ratio to that of CaIrO 3 , 0.9878( 13), but in this case the PV-structured material is softer, with separated by planar Ca-cation interlayers, a ready explanation is afforded for the relative softness of the b-axis by reduction of the interlayer spacing. However, on this basis it is difficult to explain why the c-axis is the least compressible direction in the crystal, since this axis should then be readily shortened by buckling of the planes of corner-linked octahedra, whereas to reduce the length of the a-axis must involve distortion of the PtO 6 octahedra through shortening of the O-O distances aligned parallel to the a-axis. Clearly, therefore, this view of the response to compression of post-perovskite structures in terms of layers and interlayers is too simplistic, but to properly address this question requires an accurate set of atomic coordinates as a function of pressure, which is not yet available. In connection with this discussion, it is interesting to note also that, at least in the case of CaIrO 3 , the response of the PPV-structure to heat is not simply the inverse of the response to pressure; thus, although the b-axis of CaIrO 3 expands most on heating, the expansion of the c-axis is far larger than that of the a-axis [START_REF] Lindsay-Scott | Thermal expansion of CaIrO 3 determined by X-ray powder diffraction[END_REF]Martin et al., 2007).

K 0 =
In Table 3, the ratios of the axial incompressibilities with respect to that of the c-axis, for both CaPtO 3 and CaIrO 3 , are compared to those for MgSiO 3 and Mg 0.6 Fe 0.4 SiO 3 . The low incompressibility ratio of the b-axis relative to that of the c-axis, found in both CaPtO 3 and CaIrO 3 , is similar in magnitude to that reported for MgSiO 3 at pressures and temperatures comparable to those that would obtain in the D'' region of the Earth's mantle [START_REF] Guignot | Thermoelastic properties of post-perovskite phase MgSiO 3 determined experimentally at core-mantle boundary P-T conditions[END_REF]. However, much worse agreement is found with the axial incompressibility ratios for Mg 0.6 Fe 0.4 SiO 3 at 140 GPa and ambient temperature [START_REF] Mao | Elastic anisotropy of ferromagnesian postperovskite in Earth's D'' layer[END_REF]; in this case, although the value for K a /K c agrees well with that from CaPtO 3 , neither CaPtO 3 nor CaIrO 3

give a similar value for K b /K c (though the agreement for CaPtO 3 is somewhat better than for CaIrO 3 ). A more detailed comparison of the axial compressions of CaPtO 3 with previously published results for CaIrO 3 [START_REF] Boffa Ballaran | Equations of state of CaIrO 3 perovskite and post-perovskite phases[END_REF] is shown as an inset to Figure 4.

A c c e p t e d M a n u s c r i p t

Clearly, at all pressures, CaPtO 3 is slightly less elastically anisotropic than CaIrO 3 . This difference in anisotropy may be due to distortion of the IrO 6 octahedra in CaIrO 3 , caused by the Jahn-Teller effect [START_REF] Ohgushi | CaPtO 3 as a novel post-perovskite oxide[END_REF]; the absence of this effect in CaPtO 3 may indicate that it is, therefore, a somewhat better analogue for MgSiO 3 post-perovskite at low pressure and temperature. The inset shows the f-F plot for these data, calculated using the value of V 0 , 228.10(2) Å 3 , from this fit (Table 2). The solid line shown in the inset is a weighted linear fit

with F E = 120(11)f E + 168.4(4) GPa. The value of the incompressibility, K 0 , is equal to the intercept on the y-axis of the f-F plot; the value of its first derivative with respect to pressure, K' 0 , is obtained via the relationship that the slope of the f-F plot is equal to 3K 0 (K' 0 -4)/2 [START_REF] Angel | Equations of State[END_REF]; see Section 3 for further details).

A c c e p t e d M a n u s c r i p t where A = 1904 GPa and B = 10.32(7) [START_REF] Jacobsen | Compression of single-crystal magnesium oxide to 118 GPa and a ruby pressure gauge for helium pressure media[END_REF]. Ruby measurements were made before (λ a ) and after (λ b ) each diffraction pattern was recorded and the value of Δλ was then calculated using their mean. The uncertainties in the pressure values were derived assuming that the uncertainty in the fluorescence wavelength was (λ a -λ b )/4 (i.e. that the observations corresponded to a mean value +/-two standard uncertainties); the uncertainty in the parameter B given by [START_REF] Jacobsen | Compression of single-crystal magnesium oxide to 118 GPa and a ruby pressure gauge for helium pressure media[END_REF] was also included in the calculation.

Note that in both (a) and (b) the lattice parameter values listed are probably less accurate than might be expected from the stated uncertainties, which are as reported by the Le Bail fits using GSAS; for further discussion of the importance of systematic errors in profile refinement of X-ray powder data see e.g. [START_REF] Thompson | X-ray Rietveld refinement using Debye-Scherrer geometry[END_REF].

A c c e p t e d M a n u s c r i p t For comparison with the results of Martin et al. (2007) the unit-cell volumes were also fitted to a second-order Birch-Murnaghan equation of state. The resulting values of V 0 and K 0 were 227.95(2) Å 3 and 174.0(5) GPa respectively (with K' 0 fixed at 4).

(Note that if the commonly-used ruby fluorescence pressure scale of [START_REF] Mao | Calibration of the ruby pressure gauge to 800 kbar under quasi-hydrostatic conditions[END_REF] is employed, instead of that of [START_REF] Jacobsen | Compression of single-crystal magnesium oxide to 118 GPa and a ruby pressure gauge for helium pressure media[END_REF], the volumetric equation of state parameters then become V 0 = 228.09(2) Å 3 , K 0 = 168.8(8) GPa and K' 0 = 4.16( 6)

respectively. The more recent ruby scale of [START_REF] Dewaele | Equations of state of six metals above 94 GPa[END_REF], gives values that are not significantly different from those shown in the Table above, with V 0 = 228.10(2) Å 3 , K 0 = 168.5(8) GPa and K' 0 = 4.39(6))

A c c e p t e d M a n u s c r i p t

  PANalytical X'Pert Pro diffractometer with Co Kα 1 radiation (40 kV, 30 mA), scanning over the range 10 o < 2θ < 90 o . The sample was found to contain orthorhombic CaPtO 3 (Cmcm; a = 3.12612(2) Å, b = 9.91709(6) Å, c = 7.34952(5) Å, V = 227.850(2) Å 3 ), with

  s c r i p t minor amounts of cubic Ca x Pt 3 O 4 (Pm 3 n; a = 5.7064(1) Å, V = 185.82(1) Å 3 ; Bergner and Kohlhaas, 1973) and traces of cubic Pt (Fm 3 m; a = 3.9262(2) Å, V = 60.52(1) Å 3 ; Holmes

  GPa and 4.51(6), respectively, are in good agreement with the corresponding values of 168.4(4) GPa and 4.47(4) obtained by weighted linear regression from the f-F plot

  only equation-of-state parameters for CaPtO 3 with which to compare the results of the present study are those of[START_REF] Matar | Ab initio investigation of perovskite and post-perovskite CaPtO 3[END_REF] who carried out an investigation of both PPVand PV-structured CaPtO 3 by athermal ab initio computer simulations within the local density approximation (LDA). The agreement of our experimental values {V 0 = 228.10(2) Å 3 ; K 0 = 168.2(8) GPa; K' 0 = 4.51(6)} with those from this simulation is surprisingly poor, as[START_REF] Matar | Ab initio investigation of perovskite and post-perovskite CaPtO 3[END_REF] obtained V 0 = 212.14(5) Å 3 and K 0 = 222(1) GPa, with K' 0 = 4.59(8).

  205(1) GPa and K' 0 = 4.42(5). However, this apparent difference in behaviour should probably be treated with caution. No experimental data for a PV-structured phase of CaPtO 3 have, as yet, been presented and in view of the discrepancy of the ab initio results with our experimental values for PPV-CaPtO 3 it is probably unwise to rely upon them too closely.The axial incompressibilities of CaPtO 3 are similar in form to those of CaIrO 3 . The a-axis and c-axis of CaPtO 3 have the same incompressibility (within experimental uncertainty) at ambient pressure, but the c-axis stiffens faster than the a-axis, having a value of K' 0 that is greater by almost a factor of two; in contrast, the b-axis is much softer. If the crystal A c c e p t e d M a n u s c r i p t structure of PPV-CaPtO 3 is viewed in a naive way in terms of layers of PtO 6 octahedra

Figure 1 :

 1 Figure Captions

Figure 2 :

 2 Figure 2: Observed (points), calculated (line) and difference (lower trace) X-ray powder

Figure 3 :

 3 Figure 3: Unit-cell volume of CaPtO 3 between 1.21(1) GPa and 42.50(4) GPa.

Figure 4 :

 4 Figure 4: Axial compressions, i/i 0 (where i = a, b or c unit-cell parameter), of CaPtO 3 , plotted as the ratios (i/i 0 ) 3 . The symbols denote the experimentally-observed axial and

( b )

 b Unit-cell parameters and unit-cell volume for CaPtO 3 as a function of pressure, as measured in the diamond-anvil cell at the ESRF. The data were recorded in the order shown.The numbers in parentheses are estimated standard uncertainties and refer to the least-above, the pressure values were obtained from the relative shift of the wavelength of the ruby-R 1 fluorescence line (Δλ/λ 0 ), using the equation P (GPa) = (A/B){[1 + (Δλ/λ 0 )] B -1},

  Figure 1 Click here to download high resolution image

  Figure 2A Click here to download Figure(s): figure 2a.eps

  Figure 2B Click here to download Figure(s): figure 2b.eps

  Figure 3 Click here to download Figure(s): figure 3.eps

  Figure 4 Click here to download Figure(s): figure 4.eps

Table 1 :

 1 (a) Unit-cell parameters and unit-cell volume for CaPtO 3 at ambient pressure and temperature; the numbers in parentheses are estimated standard uncertainties and refer to the least-significant digits. The value quoted for the present work is that measured in our laboratory at UCL with Co Kα 1 radiation (see Section 2).

		a(Å)	b(Å)	c(Å)	Volume (Å 3 )
	Present work	3.12612(2) 9.91709(6) 7.34952(5)	227.850(2)
	Ohgushi et al. 2008 3.12607(1) 9.91983(4) 7.35059(3)	227.942(2)
	Inaguma et al. 2008 3.1232(4)	9.912(1)	7.3459(9)	227.41(5)

Table 2 :

 2 Equation of State parameters obtained by fitting the data shown in Table1to thirdorder Birch-Murnaghan equations of state. For the unit-cell edges, the values of K 0 and K' 0 are those obtained by fitting to the cubes of the lengths of the unit-cell edges (for details see text).

		V 0 (Å 3 )	K 0 (GPa)	K' 0
	Volume 228.10(2)	168.2(8)	4.51(6)
	a-axis 3.12777(8)	195.7(8)	6.63(8)
	b-axis	9.9191(5)	123.3(5)	2.37(3)
	c-axis	7.3551(4)	192(2)	12.2(3)

Table 3 :

 3 Axial incompressibility ratios of some post-perovskite phases.

	K a /K c	K b /K c

a This work b Boffa Ballaran et al. (2007) c Guignot et al. (2007) d Mao et al. (2010); *uncertainties not given.

Acknowledgments

AL-S is funded by a postgraduate studentship from the Natural Environment Research Council. Shaun Evans is thanked for experimental assistance at ID09. We are also grateful for the helpful comments of an anonymous referee with regard to the choice of pressure scale.

A c c e p t e d M a n u s c r i p t