Ségolène Hescot

Angelo Paci

Atmane Seck

Abdelhamid Slama

Say Viengchareun

Séverine Trabado

Sylvie Brailly-Tabard

Abir Al Ghuzlan

Jacques Young

Eric Baudin

Marc Lombès
email: marc.lombes@u-psud.fr

The Lack of Antitumor Effects of o,p'DDA Excludes Its Role as an Active Metabolite of Mitotane for Adrenocortical Carcinoma Treatment

Keywords: Adrenocortical carcinoma, mitotane, o, p'-DDD, o, p'-DDA

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mitotane (o,p'DDD) represents the most effective drug for the treatment of advanced adrenocortical carcinoma (ACC), and is the only drug approved in the advanced setting [START_REF] Schteingart | Management of patients with adrenal cancer: recommendations of an international consensus conference[END_REF][START_REF] Berruti | Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF]. Mitotane is prescribed either in monotherapy or associated with platine-based chemotherapy and induces tumor response rates in up to one third of patients [START_REF] Baudin | Therapeutic management of advanced adrenocortical carcinoma: what do we know in 2011?[END_REF]. Recently, the first phase III trial performed in advanced ACC demonstrated that combination of mitotane chemotherapy with cisplatine-doxorubicine-etoposide was more effective in prolonging progression free survival and rate of objective response than the combination of mitotane and streptozotocin [START_REF] Fassnacht | Combination chemotherapy in advanced adrenocortical carcinoma[END_REF]. In addition, mitotane is also recommended in the adjuvant setting in ACC patients at high risk of recurrence [START_REF] Berruti | Adjuvant therapy in patients with adrenocortical carcinoma: a position of an international panel[END_REF][START_REF] Else | Adjuvant therapies and patient and tumor characteristics associated with survival of adult patients with adrenocortical carcinoma[END_REF].

However, the mechanism of mitotane action remains to be understood. To date, the only predictive factor of tumor response is the plasma mitotane level. Indeed, several studies have reported that a higher response rate correlates to plasma mitotane levels above 14 mg/l [START_REF] Haak | Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients[END_REF][START_REF] Baudin | Impact of monitoring plasma 1,1dichlorodiphenildichloroethane (o,p'DDD) levels on the treatment of patients with adrenocortical carcinoma[END_REF][START_REF] Malandrino | Prognostic markers of survival after combined mitotane-and platinum-based chemotherapy in metastatic adrenocortical carcinoma (ACC)[END_REF][START_REF] Wängberg | The long-term survival in adrenocortical carcinoma with active surgical management and use of monitored mitotane[END_REF][START_REF] Hermsen | Plasma concentrations of o,p'DDD, o,p'DDA, and o,p'DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study[END_REF]. Several retrospective studies have also suggested a prolonged survival in case of plasma mitotane levels above 14 mg/l [START_REF] Haak | Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients[END_REF][START_REF] Malandrino | Prognostic markers of survival after combined mitotane-and platinum-based chemotherapy in metastatic adrenocortical carcinoma (ACC)[END_REF][START_REF] Hermsen | Plasma concentrations of o,p'DDD, o,p'DDA, and o,p'DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study[END_REF] but not all [START_REF] Wängberg | The long-term survival in adrenocortical carcinoma with active surgical management and use of monitored mitotane[END_REF][START_REF] Ayala-Ramirez | Adrenocortical carcinoma: clinical outcomes and prognosis of 330 patients at a tertiary care center[END_REF]. In addition, as neurological toxicities have been found more frequent with mitotane plasma levels above 20 mg/l [START_REF] Van Slooten | The treatment of adrenocortical carcinoma with o,p'-DDD: prognostic implications of serum level monitoring[END_REF], the recommended therapeutic window is currently between 14 and 20 mg/l [START_REF] Berruti | Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF].

In a recent study, Hermsenet al. analysed for the first time the respective role of plasma mitotane level and its metabolites 1,1-(o,p'-dichlorodiphenyl) acetic acid (o,p'DDA) and o,p'DDE as predictive factors of response in advanced ACC patients. This study concluded that the plasma o,p'DDD level only was associated with tumor response while no major role for its metabolites could be retrieved, questioning the real impact of o,p'DDA as an active metabolite, as previously suggested in preclinical studies.

At the time of ingestion, only 35% of o,p'DDD is absorbed from the gastrointestinal tract [START_REF] Moy Rh | Studies of the pharmacology of o,p'DDD in man[END_REF] and o,p'DDA has been first described as the main urinary metabolite of o,p'DDD [START_REF] Sinsheimer | Identification of o,p'dichlorodiphenyl acetic acid as a urinary metabolite of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane[END_REF][START_REF] Reif | Metabolism of 1-(0-chlorophenyl)-1-(p-chlorophenyl)-2,2dichloroethane (o,p'-DDD) in rats[END_REF]. O,p'DDA is an acid derivative of o,p'DDD, obtained from two consecutive reactions of β-hydroxylation and dehydrochlorination [START_REF] Schteingart | Adjuvant mitotane therapy of adrenal cancer -use and controversy[END_REF]. The site of o,p'DDA synthesis remains unclear in humans. Martz and Straw and later Pohland et al were able to detect o,p'DDA in hepatic microsomes suggesting an hepatic metabolism of o,p'DDD [START_REF] Martz | Metabolism and covalent binding of 1-(o-chlorophenyl)-1-(pchlorophenyl)-2,2-dichloroethane (o,p,'-DDD). Correlation between adrenocorticolytic activity and metabolic activation by adrenocortical mitochondria[END_REF][START_REF] Pohland | In vitro and in vivo metabolism of a radioiodinated analog of 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,-dichloroethane[END_REF]. Quantitative analyses measuring radiolabelled o,p'DDA in various species further revealed minute amounts of o,p'DDA in adrenal mitochondria [START_REF] Martz | The in vitro metabolism of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2dichloroethane (o,p'-DDD) by dog adrenal mitochondria and metabolite covalent binding to mitochondrial macromolecules: a possible mechanism for the adrenocorticolytic effect[END_REF][START_REF] Cai | Bovine adrenal cortex transformations of mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane; o,p'-DDD] and its p,p'-and m,p'isomers[END_REF][START_REF] Cai | Metabolic activation and binding of mitotane in adrenal cortex homogenates[END_REF]. From these initial observations emerged a first hypothesis that o,p'DDA could be synthesized in the liver or the adrenals and may represent the active metabolite responsible of the cytotoxic effect observed in the adrenal cortex. Others studies suggested that an adrenal metabolic transformation of o,p'DDD into o,p'DDA could be responsible of the adrenolytic effect [START_REF] Cai | Adrenal proteins bound by a reactive intermediate of mitotane[END_REF]. Finally, the question on whether o'p,DDA is responsible for antitumor effects of mitotane in ACC patients remains totally unanswered.

We have previously described molecular effects of o,p'DDD on cell proliferation, steroidogenesis and respiratory chain activity [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF]. The aim of the present study was to investigate the exact role of o,p'DDA, in comparison with o,p'DDD. For this purpose, we first explored in vitro moleculareffects of o,p'DDA on cell proliferation, mitochondrial respiratory chain and steroidogenesis. We thus studied o,p'DDA conversion and uptake in human adrenocortical H295R and SW13 cell lines and human adrenal tissue samples.

Materials and Methods

Human adrenocortical cells and human adrenal tissues

H295R and SW13 cells were cultured in DMEM/HAM'S F-12 medium (PAA, Les Mureaux, France) supplemented with 20 mM HEPES (Life technologies, Saint Aubin, France), antibiotics (penicillin 100 IU/ml, streptomycin 100 µg/mL) and 2 mM glutamine (all from PAA). The medium for H295R cell culture was enriched with 10% fetal bovine serum and a mixture of insulin/transferrin/selenium. Both cell lines (from passage 2 to 15) were cultured at 37°C in a humidified incubator with 5% CO 2 .

Mitotane (supplied by HRA Pharma, Paris, France) and o,p'DDA (Alsachim,Illkirch-Graffenstaden, France) were solubilized in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Quentin Fallavier, France) and used at 10 to 300 µM final concentrations. The percentage of DMSO in culture medium never exceeded 0.1%. Tissues were obtained from six patients treated with mitotane. Five ACC patients were followed at GustaveRoussy, while patient 1, followed at Bicêtre Hospital, underwent bilateral adrenalectomy for an ectopic Cushing syndrome due a bronchopulmonary ACTH-secreting tumor. All patients signed an informed consent. Tissues were lysed in H 2 O using a TissueLyser apparatus (Qiagen, Courtaboeuf, France).

Steroid secretion

Steroid hormones concentrations were measured in the supernatants of H295R cultured cells using radioimmunoassays for 11-deoxycortisol (DiaSource Immunoassays, Louvain-la-Neuve, Belgium) and 17-hydroxyprogesterone (MP Biomedials, Ohio, USA) or chemiluminescent enzyme immunoassay for cortisol (Immulite 2000 XPI, Siemens Healthcare Diagnostics Products, Llanberis, UK). The intraassay coefficient of variation (CV) were 5.2% and 7.7% at 3.7 and 28.3 ng/ml, and the inter-assay CV were 11.5% and 15.1% at 5.5 and 36.9 ng/ml for 11-deoxycortisol. The intra-assay CV of 17hydroxyprogesterone radioimmunoassay were 7.8% and 8.3% at 2.7 and 13.2 ng/ml respectively and the inter-assay CV were 9.8% and 12.8% at 12.3 and 22.8 ng/ml. The intra-assay CV of cortisol assay were 6.1% and 5.2% at 33 and 85 ng/ml, and the inter-assay CV were 8.2% and 6.8% at 33 and 85 ng/ml.

Cell proliferation analysis

Cell proliferation tests were performed by using the WST1 assay (Roche, Meylan, France) according to the manufacturer's recommendations. Cells were cultured in 96-well plates and treated with 10 to 300 µM mitotane or o,p'DDA for 48 h. Optical densities were measured 4 h after addition of WST1 solution (10 µl per well) by spectrophotometry (Viktor, Perkin Elmer, Courtaboeuf, France).

Respiratory chain analysis

Respiratory chain activities were measured using spectrophotometric assays. H295R cells were treated with 50 µM of mitotane (16 mg/l), 50 µM of o,p'DDA (14 mg/l) or vehicle (DMSO) alone for 48 h and the activity of complex IV (cytochrome c oxidase, COX) was measured in a Cary 50 spectrophotometer,as previously described [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF]. The activity of Complex IV was normalized to that of citrate synthase activity, as an index of mitochondrial mass.

Reverse Transcriptase-PCR (RT-PCR) and Quantitative real-time PCR (RT-qPCR)

Total RNAs were extracted from cells with the RNeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. RNA was thereafter processed for RT-PCR as previously described [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF]. Quantitative real-time PCR (RT-qPCR) was performed using the Fast SYBR ® Green Master Mix (Life Technologies) and carried out on a StepOnePlus™ Real-Time PCR System (Life Technologies) as previously described [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF]. Standards and samples were amplified in duplicate and analyzed from six independent experiments. The ribosomal 18S rRNA was used as the internal control for data normalization. The relative expression of each gene was expressed as the ratio of attomoles of specific gene to femtomoles of 18S rRNA. The primer sequences of the genes analyzed by qRT-PCR are shown in the Supplemental Table 1.

Western Blot analysis

Total protein extracts were prepared from cells lysed in lysis buffer (50 mMTris-HCl, pH 7.5, 150 mMNaCl, 5 mM EDTA, 30 mM Na pyrophosphate, 50 mM Na fluoride, 1% Triton X-100), and 1X protease inhibitor (Sigma-Aldrich, St. Louis, MO). After protein blotting on an Odyssey nitrocellulose membrane (LI-COR, Lincoln, NE, USA), blots were incubated for 1 h at RT in a blocking buffer (5% fat-free milk in Phosphate-buffer saline PBS with 0.1 % Tween 20) before an overnight incubation at 4°C with a rabbit anti-Bcl2 antibody (1:500 dilution, Cell Signaling, Saint Quentin en Yvelines, France) and a mouse anti-α-Tubulin antibody (1:10,000 dilution, Sigma-Aldrich). After extensive washes, blots were incubated with an IRDye 800-conjugated affinity purified anti-rabbit IgG second antibody (1:15,000 dilution, Perbio Science, Brebières, France) and an IRDye 680-conjugated affinity purified anti-mouse IgG second antibody (1:15,000 dilution, Perbio Science) for 1 h at RT. After washes, proteins were visualized with an Odyssey-Fc apparatus (LI-COR). Specific signals for Bcl2 were normalized by the infrared fluorescence of α-Tubulin signals as determined by densitometry using the Image Studio software (LI-COR).

Measurements of o,p'DDD, o,p'DDA and o,p'DDE metabolites

Analyses were conducted by high performance liquid chromatography combined to an ultraviolet detection (HPLC-UV) on plasma samples of 100 µl spiked with known amounts of p,p'-DDE used as an internal standards (IS) of o,p'-DDD and o,p'-DDE andp,p'-DDAused as an internal standards (IS) of o,p'-DDA measurements. O,p'-DDD (Mitotane), o,p'-DDA and o,p'-DDE concentrations were determined through the ratio of their peak surface area to the peak surface of known concentrations of IS. The method was validated in terms of linearity, precision and accuracy, with a threshold detection limits of 2.5 mg/l, 0.1 mg/l and 0.25 mg/l for o,p'DDA, o,p'DDE and o,p'DDD, respectively.

Statistical Analysis

Results are expressed as means ± SEM of n independent replicates performed in the same experiment or from separated experiments (n). Non-parametric Mann Whitney tests were used when appropriate and differences between groups were analyzed using non-parametric Kruskall-Wallis multiple comparison test followed by a post-test of Dunn's (Prism software, GraphPad, CA). A P value of 0.05 168 was considered as statistically significant (*P<0.05; **P<0.01; ***P<0.001).

Results

Comparative analysis of o,p'DDD and o,p'DDA impact on cell proliferation

We studied the potential anti-proliferative effect of o,p'DDA on H295R and SW13 cells, testing the hypothesis that o,p'DDA might exert antitumor properties. It is well established that circulating o,p'DDA concentrations are about 5 times higher than those of o,p'DDD measured in plasma of patients treated with mitotane [START_REF] Hermsen | Plasma concentrations of o,p'DDD, o,p'DDA, and o,p'DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study[END_REF]. We thus explored functional consequences of high doses of o,p'DDA(50 to 300 µM corresponding to 14 to 84 mg/l) compared to o,p'DDD on cell proliferation using the colorimetric solution WST1 (Fig. 1). As previously reported, we confirmed that o,p'DDD inhibited proliferation of H295R cells in a dose-dependent manner, with a significant 50% decrease when cells were exposed to 100 µM for 48 h and a 95% inhibition when treated with 200 and 300 µM.

In sharp contrast, we showed that o,p'DDA, even at highest concentrations corresponding to its plasma circulating levels in patients, had no significant effect on H295R cell proliferation.

Comparative effects of o,p'DDD and o,p'DDA on respiratory chain complex IV activity

We have previously shown that mitotane significantly impaired mitochondrial respiratory chain function by selectively inhibiting enzymatic complex IV activity (cytochrome c oxidase, COX) [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF].

We thus evaluated the effects of o,p'DDA on COX activity in H295R cells by spectrophotometric assays. COX activity remained unaffected after 48 h exposure to 50 µM of o,p'DDA with a mean activity of 204 ± 13 nmol/min per mg protein, not significantly different from vehicle-treated cells (Fig. 2A). Likewise, activity of citrate synthase (CS), an index of the mitochondrial mass, was not modified in o,p'DDA-treated cells (186 ± 8 nmol/min per mg protein), as compared to vehicle-treated cells (207 ± 6 nmol/min per mg protein). When COX enzymatic activity were normalized to citrate synthase activity (Fig. 2B), o,p'DDA exposure did not modify mitochondrial respiratory chain activity whereas o,p'DDD significantly reduced COX/CS ratio, as anticipated. We also compared the effect of In response to mitotane-elicited respiratory chain inhibition, we have previously demonstrated a compensatory increase of mitochondrial biogenesis [START_REF] Hescot | Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells[END_REF]. We therefore investigated whether or not o,p'DDA was able to induce similar compensatory mechanisms. Thus, expression of the Peroxisome proliferator-activated receptor gamma coactivator 1-related Coactivator (PRC), a key regulator of mitochondrial biogenesis, was quantified by RT-qPCR (Fig. 3A). We showed that o,p'DDD exposure induces a 2-fold increase in PRC transcript levels whereas treatment with o,p'DDA has no significant effect of PRC mRNA levels, consistent with the absence of effect on respiratory chain activity described above. We also evaluated consequences of o,p'DDA treatment compared to o,p'DDD on oxidative stress and apoptosis by studying the expression of SOD2 encoding superoxide dismutase 2, an enzyme involved in reactive oxygen species (ROS)-induceddetoxification and known to be induced during oxidative stress. Steady-state levels of SOD2 messengers were significantly increased after incubation with o,p'DDD whereas they remained unchanged with o,p'DDA, suggesting that o,p'DDD but not o,p'DDA induces oxidative stress in H295R cells (Fig. 3B). We found similar results in SW13 (data not shown). At a 50 µM concentration, neither o,p'DDD nor o,p'DDA induces apoptosis as revealed by the anti-apoptotic protein B cell lymphoma 2 (Bcl2) expression using Western Blot.

However, a drastic reduction of Bcl2 expression was observed with 100 µM mitotane, such reduction was not detected upon o,p'DDA incubation, suggesting that H295R cells were unaffected by high doses of o,p'DDA(Fig. 3C).

Differential effects of o,p'DDD and of o,p'DDA on steroidogenesis

Mitotane might exert dissociated effects with distinct time-course patterns, consisting of an early inhibitory effect on steroidogenesis followed by a later cytolytic effect responsible for adrenal cortex atrophy. To explore the impact of o,p'DDA on steroidogenesis, 17-hydroxyprogesterone, deoxycortisol and cortisol concentrations were measured in the supernatants of steroid-secreting H295R cells. Exposure to 50 µM o,p'DDA failed to inhibit secretion of 17-hydroxyprogesterone (Fig. 4A) as compared to the 80% reduction observed following o,p'DDD incubation. However, when used at higher concentrations up to 300 µM,o,p'DDA slightly but significantly reduced deoxycortisol and cortisol production without affecting 17-hydroxyprogesterone secretion of H295R cells (Fig. 4B).

Expression of several genes encoding for proteins involved in steroidogenesis was studied by RT-qPCR (Fig. 4C). While 50 µM mitotane significantly induced a 80 to 95% decrease of all genes studied, o,p'DDA failed to modify expression of StAR (encoding the transporter of cholesterol into mitochondria) or CYP11A1 (encoding cholesterol desmolase) but had negligible effect on HSD3B2 (encoding 3β-hydroxysteroid dehydrogenase), CYP21 (encoding 21-hydroxylase) and CYP11B1 (encoding 11β-hydroxylase) consistent with the anti-secretory effects of higher doses of o,p'DDA.

Intracellular concentrations of o,p'DDD and o,p'DDA in human adrenocortical cells treated with mitotane

We next examined the intracellular distribution of these drugs and investigated the ability of human adrenocortical cells to uptake o,p'DDD. We also addressed the question on whether human adrenocortical H295R and SW13 cells were able to actively convert mitotane into o,p'DDA. To

O,p'DDD, o,p'DDA and o,p'DDE contents in adrenal tissues of mitotane-treated patients

In order to examine the tissue distribution of mitotane and its metabolites in humans, six samples of human adrenal tissue from mitotane-treated patients were collected. Patient 1underwent bilateral adrenalectomy for an ectopic Cushing syndrome due a bronchopulmonary ACTH-secreting tumor whereas patients 2 to 6 had ACC. Cumulative doses of mitotane, duration of treatment and plasma mitotane levels are reported in Table 1. Patient 2 had a local ACC recurrencewith an imaging work-up performed three months later showing a progressive disease according to RECIST criteria [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF]. Thus, he received mitotane for11 months and the last imaging work-up preceding surgery showed a stable disease according to RECIST criteria, defining this patient as responder to mitotane. Patient 3 received mitotane associated withcisplatin and etoposide, his last morphologic work-up before surgery showing a stable disease according to RECIST. Patient 4 and 5 are considered as non responders to mitotane.

Indeed, patient 4, presentingwith a metastatic ACC,had progressive disease according to RECIST criteria whereas patient 5 presented a local recurrence while he was treated with mitotane as an adjuvant therapy. Finally, evaluation of mitotaneresponse for patient 6 was not possible since she died shortly after surgery without undergoing imaging work-up. We quantified o,p'DDD, o,p'DDAand o,p'DDE contents in adrenal tissue homogenates measured by HPLC-UV assays (Fig. 6). Results of adrenal gland contents of mitotane and its metabolites are reported in Table 1. O,p'DDD was found 20-to 40-fold higher in normal adrenal tissue compared to ACC despite lower plasma mitotane level (3.3 mg/l for Cushing's patient vs20.03, 18.5,23.7, 14.7 and 4 mg/l for ACC patients). More importantly, in both ACC and a normal adrenal gland of mitotane-treated patients, o,p'DDA was below limit of quantification (LOQ) threshold unlike o,p'DDD.(Fig. 6).

Discussion

Mitotane remains a major therapeutic option in ACC patients and no new pharmacological strategy has been proposed for the past 30 years [START_REF] Schteingart | Management of patients with adrenal cancer: recommendations of an international consensus conference[END_REF][START_REF] Berruti | Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF]. However, only one third of patients are considered as responders and the only predictive factor of response is plasma mitotane level, which has to be between 14 and 20 mg/l, a therapeutic window reached after a mean of three months of treatment [START_REF] Baudin | Impact of monitoring plasma 1,1dichlorodiphenildichloroethane (o,p'DDD) levels on the treatment of patients with adrenocortical carcinoma[END_REF][START_REF] Mauclère-Denost | High-dose mitotane strategy in adrenocortical carcinoma: prospective analysis of plasma mitotane measurement during the first 3 months of follow-up[END_REF][START_REF] Kerkhofs | Comparison of two mitotane starting dose regimens in patients with advanced adrenocortical carcinoma[END_REF].For this reason, elucidation of mitotane mechanism of action constitutes a major field of research. Preclinical studies have suggested that o,p'DDA could be the active metabolite of o,p'DDD.

The role of o,p'DDA compound has also been recently reevaluated in ACC patients. In a first study, mean o,p'DDA plasma concentrations measured in mitotane-treated patients were found five times higher than those of o,p'DDD. However, the median time to reach maximal o,p'DDA values was found shorter than for o,p'DDD (11vs 55 days, respectively), suggesting that o,p'DDA might constitute an earlier predictor of response [START_REF] Kerkhofs | Comparison of two mitotane starting dose regimens in patients with advanced adrenocortical carcinoma[END_REF]. In a second study, o,p'DDA plasma level above 92 mg/l could be more specific than o,p'DDD above 14 mg/l in the prediction of tumor response but its mean level was not correlated with tumor response [START_REF] Hermsen | Plasma concentrations of o,p'DDD, o,p'DDA, and o,p'DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study[END_REF]. Based on the lack of convincing clinical evidence for a major role of o,p'DDA as an active antitumor metabolite of mitotane in ACC patients, we aimed at reevaluating the exact pharmacological contribution of o,p'DDA by studying its molecular effects in vitro on human adrenocortical H295R and SW13 cells and by investigating its adrenal gland distribution in mitotane-treated patients.

In order to rule out an antitumor role of o,p'DDA, we compared o,p'DDA and o,p'DDD action on cell proliferation, respiratory chain, mitochondrial biogenesis, oxidative stress, apoptosis and steroidogenesis. To the best of our knowledge, direct functional consequences of o,p'DDA have never been studied before. We first demonstrate that o,p'DDA did not exert any anti-proliferative effect on adrenocortical cells, even at high concentrations up to 300 µM (84 mg/l) corresponding to mean plasma o,p'DDA levels measured in mitotane-treated patients. Second, functional assays showed that low concentrations such as 50 µM o,p'DDA had no significant impact on mitochondrial respiratory chain activity and biogenesis, and were not able to induce H295R cell oxidative stress or apoptosis in sharp contrast to o,p'DDD, as previously described [START_REF] Lehmann | The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells[END_REF][START_REF] Poli | Morpho-functional effects of mitotane on mitochondria in human adrenocortical cancer cells[END_REF].

We quantified for the first timeo,p'DDD and its metabolites in 6 human samples of adrenal tissues.

Patients rarely receive mitotane before undergoing surgery that may explain the low number of available samples. We were unable to quantify o,p'DDA metabolite as its concentration was below LOQ, strongly suggesting a lack of in situ production and an absence of uptake of circulating metabolite by adrenal glands. Along this line, o,p'DDAwas undetectable in human adrenocortical H295R or SW13 cell supernatants and pellets after a 48 h exposure with 50 µM mitotane providing additional arguments against an active metabolism of o,p'DDD in human adrenocortical cortex.

Further studies should be performed in primary cultures of ACC to confirm the findings of the current study. Previous studies identified o,p'DDA in adrenals of bovines, dogs or rats treated with radiolabelled mitotane [START_REF] Pohland | In vitro and in vivo metabolism of a radioiodinated analog of 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,-dichloroethane[END_REF][START_REF] Martz | The in vitro metabolism of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2dichloroethane (o,p'-DDD) by dog adrenal mitochondria and metabolite covalent binding to mitochondrial macromolecules: a possible mechanism for the adrenocorticolytic effect[END_REF][START_REF] Cai | Bovine adrenal cortex transformations of mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane; o,p'-DDD] and its p,p'-and m,p'isomers[END_REF] but not all [START_REF] Cai | Metabolic activation and binding of mitotane in adrenal cortex homogenates[END_REF]. Of note, in these studies, intra-adrenal radiolabelledo,p'DDA represented less than 5% of total radioactivity. In contrast, an hepatic production of o,p'DDA has been previously reported, as demonstrated by metabolic conversion of o,p'DDD with hepatic microsomes of dogs, rats or bovines [START_REF] Martz | Metabolism and covalent binding of 1-(o-chlorophenyl)-1-(pchlorophenyl)-2,2-dichloroethane (o,p,'-DDD). Correlation between adrenocorticolytic activity and metabolic activation by adrenocortical mitochondria[END_REF][START_REF] Pohland | In vitro and in vivo metabolism of a radioiodinated analog of 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,-dichloroethane[END_REF]. Similarly, an active hepatic metabolism of o,p'DDDis likely given the mitotane-induced CYP3A4 expression in the liver [START_REF] Chortis | Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5α-reductase, explaining the need for personalized glucocorticoid and androgen replacement[END_REF][START_REF] Takeshita | Mitotane induces CYP3A4 expression via activation of the steroid and xenobiotic receptor[END_REF]. Moreover, elevated plasma mitotane levels were found to be associated with a gene polymorphism of CYP2B6 encoding a cytochrome known to catalyze the metabolism of o,p'DDT, structurally close to o,p'DDD [START_REF] D'avolio | Influence of the CYP2B6 polymorphism on the pharmacokinetics of mitotane[END_REF]. Although our study does not rule out a production of o,p'DDA outside the adrenal gland, likely in liver as previously suggested, our results strongly suggest a lack of capture by adrenal cells and therefore a low if any antitumor activity of o,p'DDA.

We confirmed an uptake of o,p'DDD by H295R cells [START_REF] Poli | Morpho-functional effects of mitotane on mitochondria in human adrenocortical cancer cells[END_REF] and demonstrated that up to 25% of o,p'DDD was measured in cell pellets, suggesting that o,p'DDD itself could be the active compound.

Interestingly, we discovered that o,p'DDD content measured into normal human adrenal gland collected after surgery of a mitotane-treated ectopic Cushing patient was much higher than that quantified in five resected ACC, while plasma circulating mitotane levels were much lower in the former patient compared to the latter (see Table 1). These observations strongly support the notion that the rate of mitotane uptake in the adrenal gland might be related to the specific transport processes active in normal adrenocortical cells while weakly expressed or absent in ACC. In ACC, o,p'DDD content was higher in adrenal tissue of the patient presenting with stable disease compared to the adrenal tissues of other patients. However, no conclusion could be drawn from this observation given the small number of samples.Altogether, our experiments bring decisive arguments to exclude that o,p'DDA is the active metabolite responsible for adrenolytic effects of mitotane.

In patients, mitotane is known to exert dual effects on adrenal cells including an early inhibition of steroidogenesis and a late destruction of adrenal tissue [START_REF] Bergenstal | Management of Addison's disease in adrenalectomized patients[END_REF]. Here, o,p'DDA may potentially exert an inhibitory effect of steroidogenesis. Indeed, although o,p'DDAup to 300 µM did not inhibit 17hydroxyprogesterone secretion, we showed a slight and significant reduction in the expression of some (HSD3B2, CYP21 and CYP11B1) but not all genes encoding steroidogenic enzymes, consistent with the weak inhibition of cortisol secretion at 300 µM o,p'DDA. Accordingly, it has been reported that the aromatic moiety of the o,p'DDD molecule could be responsible for an anti-steroidogenic effect whereas the aliphatic group of this compound could account for its cytolytic effect as previously suggested [START_REF] Schteingart | Structural requirements for mitotane activity: development of analogs for treatment of adrenal cancer[END_REF]. Indeed, o,p'DDD and m,p'DDD which display similar aromatic structures inhibited steroidogenesis while p,p'DDD that exhibits a different aromatic cycle, was unable to inhibit steroidogenesis [START_REF] Asp | Chiral effects in adrenocorticolytic action of o,p'-DDD (mitotane) in human adrenal cells[END_REF]. Thus,these findings pointed to the importance of the aliphatic group, notably the dihalogenated methane carbon which is critical for the cytotoxic activity [START_REF] Schteingart | Structural requirements for mitotane activity: development of analogs for treatment of adrenal cancer[END_REF], yet lacking in the o,p'DDA structure.

To conclude, our results indicate for the first time anabsence of adrenal cell metabolism and uptake of o,p'DDA and provide in vitro evidence that o,p'DDA is very unlikely an effective metabolite of mitotane to control adrenocortical cell proliferation and to impact mitochondrial respiratory chain activity. By contrast, we confirm major interaction of o,p'DDD with adrenal cell functions and proliferation and unambiguously demonstrate a high cellular uptake of o,p'DDD by adrenal cells.

Taken together, our results indicate that research on mitotane mechanism of action should focus on o,p'DDD.

Figures legends

  o,p'DDA and o,p'DDD on the steady-state level of mitochondrial DNA-encoded COX2 transcript (Fig. 2C). While COX2 expression was significantly reduced by more than 40% upon o,p'DDD treatment, o,p'DDA failed to modifyCOX2 mRNA levels. Similar results were obtained with nuclear DNA-encoded COX4 transcripts (data not shown). Taken together, these results clearly demonstrate that o,p'DDA was ineffective in modulating mitochondrial activity. Consequences of o,p'DDD or o,p'DDA treatment on mitochondrial biogenesis, oxidative stress and apoptosis.

  mitotane for 48 h and o,p'DDD and o,p'DDA concentrations were measured in cultured cell supernatants as well as in cell pellets by using HPLC-UV. As shown in Fig.5A, when H295R cells were exposed to o,p'DDD at a theorical concentration of 50 µM (measured concentration of 54.4 ± 6.2 µM, corresponding to a initial total amount of 325 ± 49 µg), a remaining 31.1 ± 8.7 µM concentration (200 ± 56 µg) was recovered in cell supernatants after 48 h, suggesting an approximately 40% cellular uptake of o,p'DDD. Accordingly, we were able to measure a total amount of 79 ± 5.1 µg of mitotane in cell pellets (mean ± SEM, n= 3 independent experiments) indicating that approximately 25% of o,p'DDD was actively concentrated into H295R cells while 15% of initial o,p'DDD was likely deteriorated or bound to the plate as confirmed by a cell freeculture experiment. Similar results were found with SW13 cells (data not shown). Surprisingly, under these experimental conditions, despite a substantial o,p'DDD uptake as measured above, o,p'DDA concentrations were below limit LOQ threshold in both cell homogenates and cell supernatants, indicating that H295R cells were unable to convert o,p'DDD into o,p'DDA and subsequently to secrete the acidic metabolite of mitotane.Assessment of o,p'DDA uptake in human adrenocortical H295R and SW13 cellsWe next examined the ability of H295R cells to uptake o,p'DDA as compared to o,p'DDD. As shown in Fig.5B, under experimental conditions in which H295R cells were exposed to 50 µM of o,p'DDA during 48 h, o,p'DDA concentration measured in cell supernatant was not modified (48.75 ± 0.75 µM, mean ± SEM of three independent experiments), and was below LOQ threshold in cell homogenate, suggesting that o,p'DDA was not efficiently transported into H295R cells or was not significantly retained into cell compartment.

Dose-dependent impact

  of increasing concentrations of o,p'DDD and o,p'DDA (10-300 µM) on the proliferation index of human adrenocortical H295R cells after 48 h as determined by WST1 assays. Results are expressed as mean percentage ± SEM of 6 to 18 independent determinations, values of vehicle-treated cells being arbitrarily set at 100%. O,p'DDD inhibits cell proliferation in a dosedependent manner whereas o,p'DDA has no significant impact on cell proliferation. *P<0.05 and ***P<0.001, Mann-Whitney U test.

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 5 o

 5 Figure 5

Figure 6 HPLC

 6 Figure 6 HPLC-UV analysis of o,p'DDD and o,p'DDE in human adrenal tissues: a normal adrenal (A) and an

  ). H295R cells were treated with 50 µM o,p'DDD or o,p'DDA for 48 h. Relative mRNA expression of each gene was determined using RT-qPCR. Results are means ± SEM of six different experiments performed in duplicate and are expressed as percentage of the relative expression in vehicle-treated cells, arbitrarily set at 100%. Steady-state levels of Bcl2 protein (anti-apoptotic factor) by Western Blot with anti-Bcl2 and antitubulin antibodies. H295R cells were treated with 50 or 100 µM o,p'DDD or o,p'DDA for 48 h. O,p'DDD is able to increase mitochondrial biogenesis, to induce oxidative stress and apoptosis whereas o,p'DDA fails to exert similar effects. *P<0.05 and ***P<0.001, Mann-Whitney U test.

	Figure 4
	Inhibition of 17-hydroxyprogesterone (17-OHP) in the steroid-secreting H295R cells with o,p'DDD
	but not with o,p'DDA (A). Cells were cultured with 50 µM mitotane (o,p'DDD) for 48 h and the 17-
	OHP concentrations were measured in the cell supernatants by radioimmunometric assays. Results are
	mean percentage ± SEM. of 3 independent determinations and are expressed as percentage of secretion
	under basal conditions in vehicle-treated cells; arbitrarily set at 100% (mean 17-OHP secretion was
	799 ng /48 h /mg protein).
	Inhibition of 17-hydroxyprogesterone (17-OHP), deoxycortisol and cortisol in the steroid-secreting
	H295R cells with high doses of o,p'DDA (B). Cells were cultured with 100 or 300 µM o,p'DDA for
	48 h and the steroid concentrations were measured in the cell supernatants by radioimmunometric
	assays. Results are mean percentage ± SEM. of 3 independent determinations and are expressed as
	percentage of secretion under basal conditions in vehicle-treated cells, arbitrarily set at 100% (mean
	deoxicortisol and cortisol secretion were 848 ng/48h/mg protein and 207ng/48h/mg protein
	respectively).
	Expression of genes encoding proteins involved in steroidogenesis (C). H295R cells were treated with
	50 µM o,p'DDD or o,p'DDA for 48 h. Relative mRNA expression of each gene was determined using
	RT-qPCR. Results are means ± SEM of 6 different experiments performed in duplicate and are
	expressed as percentage of the relative expression in vehicle-treated cells, arbitrarily set at 100%.
	O,p'DDD inhibits steroid production and gene expressions whereas o,p'DDA has a weaker or no
	effect. *P<0.05 and ***P<0.001, Mann-Whitney U test.

Table 1 O,p'DDD, o,p'DDA and o,p'DDE concentrations in human adrenal tissues samples of mitotane- treated patients

 1 An informed consent was obtained for each patient. O,p'DDD and its metabolites were measured in tissue homogenates using HPLC-UV analysis.

	Patients	Clinicalpresentation Treatment	Cumulative	Plasma	O,p'DDD	O,p'DDE	O,p'DDA
	(#)		duration	dose	mitotane	(µg per	(µg per	(µg per
			(months)	(g)	level	10mg of	10 mg of	10 mg of
					(mg/l)	tissue)	tissue)	tissue)
	1	Ectopic Cushing	5	225	3.3*	17.2	< LOQ	< LOQ
	2	ACC	11	720	20.03**	0.79	0.16	< LOQ
	3	ACC	21	1470	18.5**	0.21	< LOQ	< LOQ
	4	ACC	8	765	23.7**	0.44	0.11	< LOQ
	5	ACC	10	870	14.7**	0.65	< LOQ	< LOQ
	6	ACC	2	180	4*	4.1	0.6	< LOQ

LOQ: limit of quantification. * Plasma mitotane level measured two weeks before surgery ** Mean of 4 to 15 plasma mitotane levels assessed during mitotanetreatment and before surgery Supplemental

Table 1

 1 Primer sequences of the genes analyzed by RT-qPCR(5' -3')

	Gene	Amplicon	Sensesequence	Antisensesequence
	18S	71 pb	GTGCATGGCCGTTCTTAGTTG	CATGCCAGAGTCTCGTTCGTT
	StAR	121 pb	GCCACAGACTTCGGGAACAT	AGTAGCCACGTAAGTTTGGTCTTAGAG
	Cyp11A1	149pb	CGATTACCGTGGCATCCTCTA	AGGTTGCGTGCCATCTCATAC
	HSD3B2	149 pb	GGAGGAAGCCAAGCAGAAAA	CAGGTGGGTGGAGTTTGATGA
	Cyp21	180pb	GGCTGAAGCAGGCCATAGAG	CCACCCCTTGGAGCATGTAG
	Cyp11B1	99 pb	GGAGACACTAACCCAAGAGGACAT	ACGTGATTAGTTGATGGCTCTGAA
	COX 2	194 pb	TACGGCGGACTAATCTTCAA	CCGGGAATTGCATCTGTTTT
	PRC	201 pb	CCACGTACTCAGGGTT	GGTCTAGGGGCCTCTT
	SOD2	60 pb	GCAAGGAACAACAGGCCTTA	GTAGTAAGCGTGCTCCCACAC

Acknowledgements

The authors are indebted to HRA Pharma for supporting this research.

Fundings

This workwassupported by the Institut National de la Santé et de la Recherche Médicale (Inserm) and Université Paris-Sud. SH is recipient of a fellowship from HRA Pharma Laboratories (Bourse CIFRE).

Author contributions

SH, AP, EB and ML designed the study; SH, AS, AHS and SBT performed the experiments; SH, AS, AHS, SBT, EB and ML analyzed the results; AP, SV, ST and JY helped interpret the data and participated in discussions; SH, SV, EB and ML wrote the paper; all the authors have read, revised and approved the manuscript.

Declaration of interest

SH is recipient of a fellowship from HRA Pharma Laboratories (Bourse CIFRE).

The authors declare that they have no conflict of interest.


Frédéric Blanc

Elisabeth Lemaire

François Peters

Tunable fall velocity of a dense ball in oscillatory cross-sheared concentrated suspensions

The fall velocity of a dense large ball in a suspension of neutrally-buoyant non-Brownian particles submitted to horizontal oscillatory shear is studied. As the strain amplitude is increased, the velocity increases up to a maximum value before it decreases to the value that it would have in a resting suspension. The higher the frequency, the stronger the effect. The falling ball velocity can be largely increased in the presence of the oscillatory cross-shear flow. For instance, it reaches for a particle volume fraction Φ = 0.47 four times the value it has in the unsheared suspension. At small strain amplitudes, it turns out that the falling ball velocity is determined by a balance between the steady drag flow, which drives the apparent suspension viscosity toward a high value, and the oscillatory cross-shear that lessens it. A simple model is proposed to explain the experimental observations at small strain amplitude. The velocity decrease observed at larger amplitude is not completely understood yet.

Introduction

Suspensions display a wide range of rheological properties which are closely connected to the so-called shear-induced microstructure [START_REF] Morris | A review of microstructure in concentrated suspensions and its implications for rheology and bulk flow[END_REF]). In the field of suspensions of non-colloidal particles in a Newtonian liquid, the pair distribution function that quantifies the microstructure has been measured experimentally [START_REF] Parsi | Fore-and-aft asymmetry in a concentrated suspension of solid spheres[END_REF][START_REF] Rampall | The influence of surface roughness on the particle-pair distribution function of dilute suspensions of non-colloidal spheres in simple shear flow[END_REF][START_REF] Gao | Direct investigation of anisotropic suspension structure in pressure-driven flow[END_REF][START_REF] Cheng | Imaging the microscopic structure of shear thinning and thickening colloidal suspensions[END_REF]Blanc et al. 2011a[START_REF] Blanc | Microstructure in sheared nonbrownian concentrated suspensions[END_REF] and computed in simulations [START_REF] Sierou | Rheology and microstructure in concentrated noncolloidal suspensions[END_REF][START_REF] Yeo | Dynamics of concentrated suspensions of noncolloidal particles in couette flow[END_REF]. It basically shows a strongly enhanced pair probability at contact in the compression quadrant of the ambiant flow. It is known to originate in the non-hydrodynamic forces between particles and to result in a large viscosity increase together with the development of anisotropic normal stresses [START_REF] Sierou | Rheology and microstructure in concentrated noncolloidal suspensions[END_REF][START_REF] Boyer | Unifying suspension and granular rheology[END_REF][START_REF] Dbouk | Normal stresses in concentrated non-brownian suspensions[END_REF]. For example, in a shear reversal experiment [START_REF] Gadala-Maria | Shear-induced structure in a concentrated suspension of solid spheres[END_REF][START_REF] Kolli | Transient normal stress response in a concentrated suspension of spherical particles[END_REF][START_REF] Narumi | Transient response of concentrated suspensions after shear reversal[END_REF]Blanc et al. 2011b), where the microstructure is destroyed and then built again by the shear flow, the viscosity can be reduced by a factor ten or more, depending on the particle volume fraction, as the microstructure vanishes [START_REF] Bricker | Correlation between stresses and microstructure in concentrated suspensions of non-brownian spheres subject to unsteady shear flows[END_REF] before it increases again to its steady value. Such a suspension, if submitted to an oscillatory shear flow, displays a complex behavior that has been studied by several authors [START_REF] Breedveld | Shear-induced diffusion and rheology of noncolloidal suspensions: Time scales and particle displacements[END_REF][START_REF] Pine | Chaos and threshold for irreversibility in sheared suspensions[END_REF][START_REF] Bricker | Oscillatory shear of suspensions of noncolloidal particles[END_REF][START_REF] Corte | Random organization in periodically driven systems[END_REF][START_REF] Park | Rheology of oscillating suspensions of noncolloidal spheres at small and large accumulated strains[END_REF][START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF]. The viscosity at small strain amplitude is found to be much smaller than the steady shear viscosity. Again, the strong variation of the rheological properties with the strain amplitude reflects a qualitative change of the microstructure [START_REF] Bricker | Correlation between stresses and microstructure in concentrated suspensions of non-brownian spheres subject to unsteady shear flows[END_REF], and the time evolution seems to originate in the mechanical irreversibility presumably due to contact forces between particles [START_REF] Pine | Chaos and threshold for irreversibility in sheared suspensions[END_REF][START_REF] Corte | Random organization in periodically driven systems[END_REF][START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF]).

In the present paper, we focus on the slow sedimentation of a dense large ball in a concentrated suspension of neutrally buoyant non-colloidal particles in a Newtonian liquid. In an unbounded purely Newtonian liquid, the drag on the sphere is well known [START_REF] Stokes | On the effect of the internal friction of fluids on the motion of pendulums[END_REF][START_REF] Happel | Low reynolds number hydrodynamics[END_REF] and the sphere velocity is simply given as a function of the liquid viscosity η, the density of the particle ρ p , of the liquid, ρ s , the particle radius a f and the gravitational acceleration g:

v = 2 9 (ρ p -ρ s )a 2 f g η (1.1)
If the vessel boundaries influence has to be taken into account, Eq.1.1 yields an apparent viscosity that can be corrected depending on the vessel shape and the sphere position [START_REF] Happel | Low reynolds number hydrodynamics[END_REF]. The falling motion of a dense ball in concentrated suspensions has been the subject of several studies [START_REF] Mondy | Continuum approximations and particle interactions in concentrated suspensions[END_REF][START_REF] Reardon | Non-newtonian end effects in falling ball viscometry of concentrated suspensions[END_REF]. If the ball is large enough compared to the size of the particles in the suspension, and once the boundary effects are corrected, the apparent viscosity is close to the value that is measured from steady-shear rheometry. This suggests that the shear rate around the falling ball induces a microstructure in the suspension that is oriented by the local strain rate axis and that determines the viscosity felt by the ball.

Here, we present experimental evidence that the falling ball velocity increases significantly when the suspension is subjected to a uniform horizontal oscillatory shear rate, especially at low strain amplitude and high oscillation frequency, whereas the dynamic viscosity of the suspension remains roughly constant.

Experiments

Materials and experimental device

The experiments are performed in a wide-gap Couette cell made of PMMA (figure 1) mounted on a controlled stress rheometer Mars II (Haake, Thermofisher). The cylindrical cup of radius R o = 24 mm is machined into a parallelepipedic block (106 mm in height, 60 × 60 mm 2 in cross-section) and the rotor is a cylinder of radius R i = 14 mm. Transparent suspensions are made of monodisperse spherical PMMA particles (Microbeads CA30), 2a s = 31 ± 4 µm in diameter, dispersed in a Newtonian fluid (viscosity η 0 = 1.02 Pa.s) that has been designed (Cargille Laboratories) to match the density and refractive index of the particles. Two suspensions of particle volume fraction Φ = 0.40 and 0.47 are studied. Dense balls are dropped along the centerline of the gap. Their final position at the bottom of the cup is always within a f /2 of the gap center. In the following, we thus assume a constant radial position of the ball, i.e r = r c = 19 mm. We use two types of ball: stainless steel balls (specific gravity 7760 ± 80 kg.m -3 and 2a f = 1.0 mm ±10µm in diameter) and aluminium balls (specific gravity 2900 ± 30 kg.m -3 and 2a f = 1.0 mm±10µm, Goodfellow). An externally triggered camera (Basler acA2000-340km) records images of the falling ball (figure 1). Each frame (2048 px x 1088 px in size) is processed to extract the ball size and position (Blanc et al. 2011a[START_REF] Blanc | Microstructure in sheared nonbrownian concentrated suspensions[END_REF]. The apparent radius of the ball is 11 px and the accuracy in the ball center position is ±0.1 px. Since the radial position of the falling ball is constant, its vertical coordinate can be computed from its position in the frame after a necessary calibration. In order to validate the experimental set-up, steel balls are dropped in a Newtonian oil. The apparent viscosity from Eq.1.1 is approximately 10% larger than the otherwise measured viscosity. This is consistent with the influence of the cylinder walls as modeled by two infinite vertical planes 10 mm apart [START_REF] Happel | Low reynolds number hydrodynamics[END_REF]. No influence of the oscillatory shear rate is observed.

Rheological characterization

Local viscosity measurements in the wide-gap Couette cell (Blanc et al. 2011c) show a slightly shear thinning behavior of the suspensions in the range of shear rate applied in this study, namely 10 -3 s -1 γ 1 s -1 . The viscosity at Φ = 0.40 (resp. 0.47) and γ ∼0.1 s -1 is η s = 12.8 Pa.s (resp. 58.9 Pa.s) and the constitutive law is σ ∼ γ0.975 (resp. σ ∼ γ0.875 ).

Dynamic rheological measurements performed either using the wide-gap rheometer at the same time as the falling ball experiments (figure 2a) or in a parallel-plate torsional flow (figure 2b) are in good agreement. Since the out-of phase-viscosity is always quite small, only the magnitude of the complex dynamic viscosity |η * | is considered in the following. It does not vary much in the strain amplitude range of interest (10 -4 γ 0 1) and it does not depend on frequency at all in the range (0.2 Hz f 20 Hz). As a consequence, the strain amplitude γ 0 at the middle of the gap is computed from the angular amplitude of the rotor assuming a Newtonian behavior of the suspension. The time evolution of the viscosity deserves a few comments: after a very short transient (one or two cycles) following the onset of the oscillations, |η * | goes to an initial value that is much smaller than the steady shear viscosity at γ ∼ 0.1 s -1 . Then it changes slowly, but the variations do not exceed 5% of the initial viscosity for Φ = 0.40 (resp. 10% for Φ = 0.47). This behavior is in qualitative agreement with previously published short term viscosity measurements [START_REF] Breedveld | Shear-induced diffusion and rheology of noncolloidal suspensions: Time scales and particle displacements[END_REF][START_REF] Park | Rheology of oscillating suspensions of noncolloidal spheres at small and large accumulated strains[END_REF][START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF]. The low dynamic viscosity has been attributed to the development of a particular shear-induced microstructure [START_REF] Corte | Random organization in periodically driven systems[END_REF][START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF].

Results

Falling motion in suspensions at rest

When a ball is dropped in a resting suspension, the velocity undergoes the same type of transient as in the Newtonian oil (figure 3), as already reported [START_REF] Reardon | Non-newtonian end effects in falling ball viscometry of concentrated suspensions[END_REF], before it reaches a steady value v 0 . The apparent viscosity from Eq.1.1 is within 10% of that is measured using a rheometer at the same steady shear rate as estimated by γsed = v 0 /a f ∼ 0.6 s -1 . Since (R o -R i )/a f = 20, the bounding wall effect is expected to be the same as in a Newtonian liquid [START_REF] Mondy | Continuum approximations and particle interactions in concentrated suspensions[END_REF]). An important point is that this steady velocity depends only slightly on the initial suspension microstructure. The same measurement performed after the suspension was oscillatory presheared (figure 3) yields a steady velocity only 5% (resp. 10%) larger at Φ = 0.40 (resp. Φ = 0.47), whereas the dynamic viscosity during oscillation is approximately 0.35 (resp. 0.12) times the viscosity in steady shear flow. At last, even though we did not perform extensive measurements, the falling ball velocity seems to be the same whether the measurement is done when the suspension is continuously sheared or at rest. This observation has been made for a steady shear rate equal to γ = 0.35 s -1 , i.e. γa f /v 0 ∼ 0.6, in the Φ = 0.40 suspension. All these observations hold also for the falling motion of the aluminium balls. It confirms that the microstructure induced by the sphere motion itself is mostly responsible for the high viscosity felt by the sphere in steady motion.

Falling motion in oscillatory cross-sheared suspensions

An oscillatory torque is now applied and the ball vertical position z, together with the shear strain amplitude γ 0 , is measured one time per cycle. In all reported experiments, the inertia of the suspension and of the falling ball can be neglected. Figure 3 displays the falling ball velocity as oscillation alternates with rest periods. As the oscillation is applied, the sphere velocity increases during typically two cycles to a nearly constant value, v, while the viscosity probed by the rheometer reaches a steady value, shown in figure 2. As the oscillation is stopped, the velocity decreases to its initial value v 0 . The normalized fall velocity v/v 0 for the suspension with volume fraction Φ = 0.40 is displayed in figure 4a for steel and aluminium balls as a function of the strain amplitude γ 0 for different values of the frequency. Each point corresponds to the falling ball velocity averaged over the plateau after transient (figure 3), resulting in a standard deviation that roughly corresponds to the size of the symbols. As expected from Eq.1.1 v 0Steel /v 0Alu ≈ 4. The striking feature is here that the velocity largely depends on the frequency and the strain amplitude of the external shear flow, whereas the suspension viscosity measured by the rheometer hardly changes with both parameters. The same results are displayed as a function of the ratio of the imposed shear rate γ = 2πγ 0 f to the shear rate induced by the falling ball motion v/a f (figure 4b). With this scaling, all data collapse onto one single curve at small strain amplitude. In that range, the velocity seems to be determined by the competition between the falling motion of the sphere that builds the usual microstructure around it, and the external oscillatory shear rate that would destroy it. However, as the strain amplitude increases beyond typically γ M ∼ 0.2 -0.3, the former scaling does not hold anymore. The velocity decreases and reaches at a strain amplitude around γ m ∼ 0.9 the value that it would have in a resting suspension. This strong decrease cannot be explained by the quite weak variation of the dynamic viscosity with the strain amplitude (figure 2). The same qualitative features are evidenced in the Φ = 0.47 suspension with γ M ∼ 0.15 and γ m ∼ 0.6 (figure 5).

Model and discussion

To get a deeper insight into the small amplitude behavior, we build a simple model, where the apparent viscosity obeys a relaxation equation that involves two competing mechanisms:

dη app dt = - v a f η app -η max γ c1 - 2πf γ 0 γ c2 (η app -η min ) (4.1)
where v and v 0 are respectively related to η app and η max through η app /η max = v 0 /v (Eq.1.1). The first term is related to the building of the suspension microstructure around the falling ball that drives the apparent viscosity towards the value η max , which is deduced from the velocity v 0 in the resting suspension. In analogy with shear reversal experiments (Gadala-Maria & Acrivos 1980; [START_REF] Kolli | Transient normal stress response in a concentrated suspension of spherical particles[END_REF][START_REF] Narumi | Transient response of concentrated suspensions after shear reversal[END_REF]Blanc et al. 2011b), where the viscosity is controlled by the total strain experienced by the suspension, a characteristic strain γ c1 together with the typical shear rate around the falling ball v/a f determines the relaxation rate (see supplementary material). The characteristic relaxation length z c = a f γ c1 is roughly estimated from the transient velocity after the oscillation is stopped (figure 3). The characteristic strain γ c1 ≈ 3.5 (resp. 2.3) at Φ = 0.40 (resp. Φ = 0.47) is in qualitative agreement with shear reversal experiments with the same suspension (Blanc et al. 2011b, and supplementary material). The second relaxation term of Eq.4.1 drives the apparent viscosity to the lower value η min . The cor-responding relaxation rate is taken proportional to the imposed shear rate 2πf γ 0 that is responsible for the modification of the microstructure. The ratio γ c2 /γ c1 , together with η min /η max , is deduced from the best fit of the experimental measurement to the steady solution of Eq.4.1 (figures 4b & 5b). γ c2 is equal to 5.3 (resp. 2.2) at Φ = 0.40 (resp. Φ = 0.47). γ c1 and γ c2 are of the same order of magnitude, suggesting that both building and destruction of the microstructure are driven by the strain experienced by the suspension around the sphere. The ratio η min /η max is the inverse of the larger relative velocity v/v 0 that the model could predict. As expected, the computed values (η min /η max = 0.52 at Φ = 0.40 and η min /η max = 0.22 at Φ = 0.47) are quite close to the relative dynamic viscosity shown in figure 2. As shown in supplementary material, the parameters found above allow us to compute the transient response at the inception and the end of the oscillations, and the agreement is quite satisfactory. While this simple model seems to catch the mechanisms that control the falling ball velocity at low strain amplitude, it is unable to explain the decrease of the velocity as the amplitude grows further. The decrease of the falling ball velocity seems to be controlled by the strain amplitude: for a given volume fraction, the velocity reaches its maximum value at approximately the same strain amplitude, γ M , whatever the frequency, and decreases to v 0 for approximately the same amplitude γ m . This behavior deserves a few comments. First, even though the viscosity measured using the rheometer does not vary in a large extent over the amplitude range of interest, one cannot but notice that the increase of dynamic viscosity occurs from approximately γ M (figure 2). In addition, this typical strain amplitude determines approximately the minimum of the viscosity in a shear reversal experiment performed with the same suspension (Blanc et al. 2011b, and supplementary material). This set of observations suggests that the effect of the imposed shear on the suspension microstructure changes qualitatively as the strain amplitude exceeds γ M . In recent papers, such a qualitative change was evidenced [START_REF] Pine | Chaos and threshold for irreversibility in sheared suspensions[END_REF][START_REF] Corte | Random organization in periodically driven systems[END_REF], i.e. the development of long term irreversibility and diffusion for strain amplitudes larger than a critical value γ c . Below γ c , oscillations drive the system into a so-called absorbing state where the particles do not longer undergo mutual collisions. The time necessary to reach this state is an increasing function of the amplitude and seems to diverge at γ c [START_REF] Corte | Random organization in periodically driven systems[END_REF]. In recent rheological experiments [START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF]) the short term evolution of the rheological properties toward a steady oscillatory state showed the same slowing down, which was attributed to the same origin. [START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF] found γ c = 1.22 at Φ = 0.40 and γ c = 0.67 at Φ = 0.5, which is close to the values that we measure for the strain amplitude γ m necessary for the velocity to decrease to v 0 , namely γ m ∼ 0.9 (resp. 0.6) at Φ = 0.40 (resp.Φ = 0.47). In addition, the amplitude where the transient time measured by [START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF] starts increasing compares well with γ M in our experiment. Even though, in our case, the suspension around the ball cannot reach the absorbing state, since it is constantly structured by the drag flow induced by the falling motion, the decrease of the velocity above γ M could be explained by the weaker efficiency of the external shear flow in breaking the structure, akin to the increasing time necessary to drive the suspension toward a steady oscillatory behavior.

Conclusion

In this paper, we have shown that it is possible to increase significantly the fall velocity of a large dense ball in a concentrated suspension by applying an oscillatory smallamplitude horizontal cross-shear strain. The higher the particle volume fraction, the greater the effect. The falling motion of the dense ball can not be predicted by the sus-pension dynamic viscosity alone since the latter hardly changes with strain amplitude and frequency. Apparently, the motion of the ball changes the microstructure in its vicinity, resulting in an apparent viscosity that strongly depends on the oscillatory cross-shear flow characteristics. At small amplitude, the ball velocity increase is determined by the competition between the falling motion of the sphere that builds the usual microstructure around it, and the external oscillatory shear rate that breaks it. The velocity decrease that occurs at larger amplitude is not completely understood yet and seems to be connected to the increasing time that is necessary for the particles to rearrange under the influence of the oscillatory shear [START_REF] Corte | Random organization in periodically driven systems[END_REF][START_REF] Lin | Short-term and long-term irreversibility in particle suspensions undergoing small and large amplitude oscillatory stress[END_REF].

More generally, this study adresses the issue of the suspension response to the superposition of two orthogonal shear rates. This has proven fruitfull in the field of viscoelastic fluids [START_REF] Van Den Brule | Effects of fluid elasticity on the static and dynamic settling of a spherical particle[END_REF][START_REF] Padhy | Simulations of a sphere sedimenting in a viscoelastic fluid with cross shear flow[END_REF] or particle suspensions in a yield-stress fluid [START_REF] Ovarlez | Shear-induced sedimentation in yield stress fluids[END_REF]. While this issue has been rarely considered in the field of particle suspensions in Newtonian liquids [START_REF] Barral | Superposition d'écoulements orthogonaux dans des fluides complexes: mise en place de l'expérience, application aux suspensions et aux fluides à seuil[END_REF][START_REF] Hanotin | Vibration-induced liquefaction of granular suspensions[END_REF], it provides an opportunity to probe the influence of the microstructure as it is not simply imposed by a steady simple shear rate, but can be tuned using the oscillatory shear flow. In our experiments, the actual flow around the ball is probably complicated, and we think that further experiments in a more controlled rheometric cross-shear flow would provide valuable information on the connection between microstructure and rheology.

The possibility to tune the settling velocity of a non-neutrally buoyant falling ball in suspensions should have some applications in problems involving suspension degassing. Such an effect might also be involved in soil liquefaction during earthquakes.

The authors gratefully acknowledge Laurent Lobry for fruitfull discussions.

  Figure 1: Experimental device.

Figure 2 :

 2 Figure 2: Dynamic viscosity |η * | normalized by the steady viscosity at γ ∼ 0.1 s -1 against the strain amplitude for different frequencies of strain oscillation. Measurements performed : (a) in the wide-gap Couette cell (Φ = 0.40 and 0.47); (b) in a parallel-plate geometry (Φ = 0.40).

Figure 3 :Figure 4 :

 34 Figure 3: Fall velocity of a steel ball as a function of vertical position. Φ = 0.40. (--) Velocity in suspension at rest after steady preshearing [shear rate ∼ 0.1 s -1 during 60 s]; (-) Velocity in suspension at rest after the same steady preshearing followed by 6000 cycles of oscillation (f =5 Hz, γ 0 ≃ 0.33); (•) Velocity during a sequence of oscillations (f =5 Hz) interspersed with rest times.

Figure 5 :

 5 Figure 5: (a) Relative velocity increase as a function of strain amplitude. (b) Relative velocity increase as a function of reduced shear rate; same legend as in (a). Solid line: model. Φ = 0.47.

† Email address for correspondence: Francois.Peters@unice.fr