N Possémé

T David

T Chevolleau

M Darnon

Ph Brun

M Guillermet

J P Oddou

S Barnola

F Bailly

R Bouyssou

J Ducote

R Hurand

C Vérove

O Joubert

Porous SiOCH integration: Etch challenges with a trench first metal hard mask approach

The use of a metallic hard mask approach for porous dielectric film integration implies for patterning processes, different difficulties like dimensional control, bottom line roughness and residue formation or chamber conditioning. In this paper we propose to present these issues and associated solutions for p-SiOCH integration in dual damascene structure using a trench first metallic hard mask approach.

I.

Introduction

The choice of copper/ultra low-k interconnects architecture is one of the key building blocks for integrated circuit performance, process manufacturability and scalability. Many low-k materials have been proposed as silicon containing (silica-based and silsesquioxane) or non silicon containing materials [START_REF] Shamiryan | materials today[END_REF]. Finally, from the 90 nm technological node, the semiconductor manufacturers opted to go with methyl groups (Si-CH 3 ) containing organosilicate materials close to the well known silicon dioxide (SiO 2 ) [START_REF] Grill | proceedings IITC[END_REF]. The SiOCH trench patterning can be achieved using different hard mask strategies (metallic or organic hard masks), all presenting different advantages and drawbacks in terms of selectivity, faceting, etc. [START_REF] Darnon | [END_REF]. But from the 45nm interconnect technology node, with porosity introduction into SiOCH materials, the metallic hard mask strategy appears to be a promising integration scheme compared to a full organic hard mask approach. Indeed with porosity introduction, the dielectric film presents a high sensitivity upon fluorocarbon (FC) etching [4] and ashing plasma exposures [5] which can lead to dielectric constant increase and reliability degradations. In this context, the metallic hard mask integration provides a greater compatibility with porous dielectric materials than the organic hard mask approach since no ashing step is required. However, in all cases, etching processes involved in dual damascene structures fabrication generate serious issues.

In this paper we propose to present and discuss the porous SiOCH (p-SiOCH) integration difficulties using a trench first metallic hard mask integration scheme (TFMHM) with the following stack: starting from the silicon substrate: a silicon-carbonitride layer deposited by plasma-enhanced-chemical vapor deposition (PECVD SiCN) / porous carbon-doped silicon-oxide (PECVD p-SiOCH, 25% porosity, k = 2.5) / PECVD silicon dioxide (SiO 2 ) / titanium nitride deposited by physical vapor deposition (PVD TiN). We will focus on problems encountered during metal hard mask (MHM), via and line etching steps with associated solutions. The control of line dimensions is fine tuned during this step by playing with the different plasma parameters (like RF bias voltage) or TiN over-etch time. But the low etching resistance to resist during the TiN etching combined with the shrink in resist thickness at each new technological node makes this step very critical. The solution to overcome this issue is either to develop new metallic hard mask etch chemistries, like methane-based chemistries allowing a two-times selectivity improvement to the resist (Fig. 1(c)), or to transfer the line into the metallic hard mask using a tri layer approach (not shown here). In this later case, the line dimension is fine tuned by playing on the CF 4 /O 2 ratio during the SiARC opening. Then the organic planarized layer (OPL) and TiN layers are opened using NH 3 -and chlorine-based chemistries, respectively. After TiN opening, the remaining resist (for the conventional approach) or OPL (for the tri-layer approach) are removed using an O 2 -based (Fig. 1(f)) or He/H 2 /O 2 downstream plasmas, respectively.

II. Partial via opening

The shallow hard mask topography, is then planarized with an organic BARC and the via lithography is performed (Fig. 2(a)).

I. Metal hard mask opening

A thin metallic hard mask layer is deposited on the top of a dense dielectric layer (SiO 2 ) which encapsulates the underlying ultra low-k material. The trench photolithography is performed using a positive tone photoresist coupled with a bottom antireflective layer (BARC) (Fig. 1(a)). The BARC and titanium nitride layers are etched using a chlorinebased chemistry in a dedicated transformer-coupled plasma (TCP) (Fig. 1(b)).

III.1 Bottom line roughness and hard mask faceting

The two first problems encountered during the line-via etching, using low polymerizing FC chemistry at substrate temperatures above 50°C, are the hard mask faceting in via-line misaligned structures (Fig. 3(a)) and bottom line roughness formation (Fig. 3(b)). In the first case, it is difficult to control the line-via dimensions which can lead to short defects while the second issue degrades the time dependent dielectric breakdown.

III.1.1 Mechanism of TiN etching and bottom line roughness formation

Previous studies on the p-SiOCH etching in fluorocarbon-based plasmas using TiN as a hard mask reported that the TiN etching is mainly driven by an ion-assisted chemical etching mechanism, and that the etch rate, increasing with temperature, is controlled by the fluorine concentration in the plasma gas phase, and the ion bombardment density and energy [START_REF] Darnon | [END_REF]. The p-SiOCH roughness formation is explained by different mechanisms like intrinsic roughness or micromasking (coming from reactor wall conditioning or FC species), increased by the low volatility compounds (TiF x ) coming from the sputtered hard mask. The via is partially etched into the p-SiOCH material using a fluorocarbon (FC) chemistry in a capacitive coupled plasma (CCP) etcher (Fig. 2(b)). Similarly to MHM etching, the problem during this step is the low selectivity to the resist which becomes a major problem to control the via dimensions. Furthermore, a highly physical etching step used to sputter the TiN in the misaligned vias can lead to arcing in the plasma chamber. The solution is the use of a tri layer approach previously described. In this case the via dimension is fine tuned by the CF 4 /O 2 ratio during the SiARC opening then the OPL and dielectrics (SiO 2 +p-SiOCH) are open using NH 3 -and FC-based chemistries, respectively. The remaining resist or OPL are removed by an O 2 (Fig. 2(c)) or NH 3 plasma, respectively.

III. Via-line etching

The last step in the p-SiOCH via-line patterning using a TFMHM approach is the via-line etching processed with the same fluorocarbon (FC) based chemistry followed by the etchstop layer (SiCN) opening with a CH 3 F/CF 4 /N 2 /Ar chemistry. But this step must be optimized in order to overcome the different issues that we will present.

III.1.2 Solutions

The solution to limit the metallic hard mask faceting during line-via etching is to develop process at temperature lower than 50°C to reduce TiN etching reaction (Fig. 3(e)). However, this can lead to profile distortions [START_REF] Darnon | [END_REF] and bottom line roughness increase due to (in both cases) the condensation of non-volatile TiF x compounds coming from the TiN mask sputtering. Finally, using a metallic hard mask approach, the best way is to develop etching processes at substrate temperature above 50°C (to limit sputtered metal species condensation leading to micromasking) with a highly polymerising chemistry to protect the MHM (Fig. 3(d)) and prevent the p-SiOCH surface roughening (Fig. 3(c)) [6].

III.2 Residues formation

The metallic hard mask approach suffers also from specific issues such as the growth of metallic residues on top of the mask after dielectric materials etching in fluorocarbonbased plasmas (Fig. 4(b)). These residues grow with air exposure time and are not removed after conventional wet cleaning steps (basic solution + 0.1%HF) (Fig. 4(b)). They prevent a good metal barrier deposition conformity, which generates via or line opens (Figs. 4(d) and 4(e), respectively) after chemical mechanical polishing (CMP) and strongly impacts the electrical yield (Fig. 4(c)).

III.2.1 Mechanism

The mechanism of formation was attributed to fluorine species reacting with the titanium nitride and catalyzed by air moisture. More precisely, fluorine species present on the TiN surface after plasma exposure react with air moisture from the atmosphere leading to HF formation. HF reacts with the partially oxidized TiN surface and forms metallic salts through the following sequence [7]:

4F + 2H 2 O -> 4HF + O 2 HF + TiO -> TiOFH
Therefore, the solution to avoid the metallic residue formation is to act on the acid formation by either removing the fluorine or to avoid the reaction with the air moisture.

III.3 Dielectric line undulation

With the constant scaling down of dimensions, etching process steps are facing new issues. Indeed, with patterning of sub-50 nm porous SiOCH trenches using a metallic hard mask approach, a line undulation phenomenon, also called line wiggling, is observed (Fig. 5(b)). Mechanical simulations performed using the ANSYS™ code [10] show this line undulation is due the compressive residual stress of the metallic hard mask (Fig. 5(a)) combined with the p-SiOCH film thickness (not shown). The Young modulii of the metallic hard mask and the p-SiOCH also play a role on the wiggling formation but in second order compared to the previous parameters (not shown). Therefore, one solution to continue the porous material integration with a metallic hard mask for advanced interconnect technology nodes is to develop TiN materials with lower residual stresses and thicknesses (Fig. 5(c)). In addition, the effect of the p-SiOCH film thickness on line undulation will be minimized for advanced technology node, since the p-SiOCH thickness continuously decreases (aspect ratio almost constant).

IV. Chamber conditioning

The use of a TFMHM has also a strong effect on process stability and reproducibility. Indeed, with this metal hard mask approach, a drift of the line etching processes (etch rates, uniformity) and lower mean time between cleans (compared to the use of an

III.2.2 Solutions

Different in situ post-etch plasma treatments such as hydrogen-and methane-based plasmas are efficient to avoid residue formation after one day air exposure (Fig. 4(a)), strongly improving the yield (Fig. 4(c)). This efficiency was explained by the fact that hydrogen-based plasmas act on the kinetic of residue formation by removing the fluorine concentration on top of the titanium nitride layer. With methane-based chemistries, the formation of a carbon and nitrogen based passivation layer on top of the titanium nitride layer prevents the reaction with air moisture [8]. The compatibility and benefit (on reliability) of methane PET addition with porous SiOCH integration using TFMHM has been demonstrated for 45nm interconnect technology node [9]. A solution to delay this chamber defectivity is to heat the top electrode (above 100°C) to limit Ti species redeposition and thus silicon micro-masking (Fig. 6(b)). A second possibility is to clean up the reactor walls after each wafer being etched by developing appropriate plasma clean processes. For instance, Chevolleau et al. propose to use a SF 6 plasma to remove the fluorocarbon layer and Ti based species followed by an O 2 flash plasma to clean up the chamber walls from the remaining carbon [11].

V. Conclusion

Major difficulties of the p-SiOCH material integration using a TFMHM integration have been presented from the etching point of view. The first problem encountered during the hard mask and partial via etching is the low selectivity to resist leading to dimensional control issue. The use of a tri layer approach can solve this problem. While during vialine etching we are dealing with hard mask faceting, bottom line roughness and residue formation or line undulation. In this case the use of TiN hard mask with a low residual stress and thickness but also the development of highly polymerizing chemistries followed by in situ post etch treatment addition at high substrate temperature (above 50°C) are required. Finally, with the use of a metallic hard mask, reactor cleaning strategies must be revisited to allow better process stability and reproducibility.

Fig. 1 :

 1 Fig. 1: Sequence of metallic hard mask opening from line lithography (a), BARC and hard mask opening with either chlorine only (b) or with methane addition (c) chemistries followed by the resist removal with an O 2 based plasma (d).

Fig. 2 :

 2 Fig. 2: Sequence of partial via etching from via lithography (a), BARC/SiO 2 /p-SiOCH etching in FC based plasma (b) followed by resist removal in O 2 plasma (c)

Fig. 3 :

 3 Fig. 3: Top view of problems encountered during via/line etching using a low polymerizing chemistry at substrate temperature above 50°C: hard mask faceting (a), bottom line roughness (b). High polymerizing chemistry at substrate temperature above than 50°C allows to solve both issues (c) while developing low polymerizing chemistry at substrate temperature lower than 50°C improves TiN faceting (d).(c)

Fig. 4 :

 4 Fig. 4: Study of residue formation with (a) and without (b) in-situ post etch treatment addition after etching and one day air exposure. Effect on yield as a function of the waiting time between etch and wet steps (c) with the formation of defects like via (d) and line (e) opens.

Fig. 5 :

 5 Fig. 5: Comparison of the line wiggling formation between simulation prediction (a) and experimental results as a function of the line dimension for the stack 20nm SiCN/ 290 nm p-SiOCH/ 40nm SiO 2 with high (b) and low (c) TiN residual stress.

Fig. 6 :

 6 Fig. 6: Observation of (a) non heated and (b) heated (>100°C) top electrodes after more than 500 Rf hours processes.


S Wilson'

Sophie Disease

MD Heissat

Amélie Harel

Khémary Um

MD Anne-Sophie Brunet

MD, PhD Valérie Hervieu

MD Olivier Guillaud

MD Jerome Dumortier

MD, PhD Alain Lachaux

PhD Elisabeth Mintz

PharmD, PhD Muriel Bost

Dr Sophie Heissat
email: sophie.heissat@chu-lyon.fr

EVALUATION OF THE ACCURACY OF EXCHANGEABLE COPPER AND RELATIVE EXCHANGEABLE COPPER (REC) IN A MOUSE MODEL OF

Keywords: Wilson's disease, IHCu: Intrahepatic copper, CuEXC: exchangeable copper, REC: relative exchangeable copper, Cus: total serum copper, LEC: Long-Evans Cinnamon, WT serum alanine aminotransferase, AST: serum aspartate aminotransferase, Exchangeable copper, REC, Atp7b -/-mice, diagnostic test, cirrhosis

Wilson's disease (WD) is caused by mutations in the ATP7B gene responsible for a toxic copper overload mainly in the liver and the central nervous system. Phenotypic heterogeneity may challenge the diagnostic confirmation. Exchangeable copper (CuEXC) has recently been proposed as a new marker of WD, and its ratio to the total serum copper (Cus), Relative Exchangeable Copper (REC=CuEXC/Cus), as a diagnostic marker. This study aimed to investigate whether this could be confirmed in Atp7b -/-mice, an engineered WD animal model. Atp7b -/-(n=137) and wild type (WT; n=101) mice were investigated under the same

conditions at 6-8, 20, 39, or 50 weeks of age. Twenty-four Atp7b -/-mice received Dpenicillamine treatment from 39 to 50 weeks of age. Serum and liver [histology and intrahepatic copper (IHCu)] data were evaluated. In the WT group, all serum and liver data were normal. Atp7b -/-livers developed a chronic injury from isolated moderate inflammation (6-8 weeks: 16/33=48%) to inflammatory fibrosis with cirrhosis (50 weeks: 25/25=100% and 16/25=64% respectively). Cus and CuEXC increased until week 39, whereas IHCu and REC were stable with increasing age and much higher than in WT mice (mean±SD: 669±269 vs.

13±3 µg/g dry liver and 39±12 vs. 11±3%, respectively). A threshold value of 20% for REC provided a diagnostic sensitivity and specificity of 100%, regardless of sex, age, or the use of D-penicillamine. Eleven weeks of 100 mg/kg D-penicillamine reduced liver fibrosis (p=0.001), IHCu (p=0.026) and CuEXC (p=0.175). In conclusion, this study confirms REC as a WD diagnostic marker in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment. ATPase [START_REF] Bull | The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene[END_REF][START_REF] Tanzi | The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene[END_REF]. In humans, mutations in ATP7B lead to decreased biliary copper excretion and reduced incorporation into ceruloplasmin. Consequently, copper accumulates in hepatocytes and the brain, resulting in severe hepatic and/or neurological impairment [START_REF] Ala | Wilson's disease[END_REF]. The diagnosis of WD is challenging since delay or failure to diagnose can lead to chronic copper accumulation and irreversible damage [START_REF] Medici | Liver transplantation for Wilson's disease: The burden of neurological and psychiatric disorders[END_REF][START_REF] Roberts | Diagnosis and treatment of Wilson disease: an update[END_REF][START_REF] Walshe | Cause of death in Wilson disease[END_REF]. Currently WD diagnosis is based on a combination of clinical (hepatic and neurological signs, Kayser-Fleischer ring), biological (low ceruloplasmin, low total serum copper, high urinary copper excretion), and radiological findings (brain MRI) [START_REF] Roberts | Diagnosis and treatment of Wilson disease: an update[END_REF].

Clinical and biological phenotype may be heterogeneous in WD patients. Intrahepatic copper (IHCu) measurement can help diagnose WD (≥ 250 µg/g dry weight), but liver biopsy is an invasive approach. In addition, increased IHCu ( 250µg/g dry weight) can also be found in [START_REF] Roberts | Diagnosis and treatment of Wilson disease: an update[END_REF][START_REF] Ferenci | Diagnostic value of quantitative hepatic copper determination in patients with Wilson's Disease[END_REF]. Molecular genetic tests can confirm the diagnosis, but they are not available in all countries [START_REF] Schilsky | Genetic testing for Wilson disease: availability and utility[END_REF]. Thus, a rapid and reliable diagnostic marker is needed.

A C C E P T E D M A N U S C R I P T other cholestatic diseases
In a seminal study El Balkhi et al. proposed exchangeable copper (CuEXC) and relative exchangeable copper (REC) as a new biological test for WD diagnosis [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF]. CuEXC corresponds to the labile fraction of serum copper that is not complexed to ceruloplasmin. It is thought to reflect blood and tissue copper and to increase in case of overload. REC is the ratio of the labile fraction to the total serum copper (Cus), REC = CuEXC/Cus. In a patient with copper overload, REC >18% has a specificity and sensitivity of 100% for the diagnosis of WD [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF][START_REF] Trocello | Relative exchangeable copper: a promising tool for family screening in Wilson disease[END_REF][START_REF] Guillaud 0 | Relative exchangeable copper: a valuable tool for the diagnosis of Wilson disease[END_REF]. Recently, REC and CuEXC have been evaluated in Long-Evans Cinnamon (LEC) rats, another animal model of WD [START_REF] Schmitt | Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat[END_REF]. REC appeared as a reliable WD diagnostic marker and high CuEXC levels correlated with acute liver failure. Thus, the authors have suggested that CuEXC could be used as a biomarker of disease progression. However, these data were obtained in only one animal model that mostly presents acute hepatitis [START_REF] Kasai | Clinicopathological studies of LEC rats with hereditary hepatitis and hepatoma in the acute phase of hepatitis[END_REF][START_REF] Wu | The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene[END_REF] and the capacity to follow disease progression under copper chelating treatment has not been investigated.

The aim of the present study was therefore to evaluate the usefulness and reliability of REC for diagnosis and CuEXC as a marker of disease course and treatment in a mouse model of WD. We used Atp7b -/-mice from a genetically engineered rodent strain that was generated in 1999 [START_REF] Burkhead | Systems biology approach to Wilson's disease[END_REF][START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF][START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF]. As seen in human WD, these mice have hepatic intracellular copper accumulation, low serum ceruloplasmin activity, increased copper excretion in urine and chronic histological liver features of WD [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF][START_REF] Gray | Urinary copper elevation in a mouse model of Wilson's disease is a regulated process to specifically decrease the hepatic copper load[END_REF].

Material and methods

A

C C E P T E D M A N U S C R I P T

Mice, animal care, and experimental procedure

All mice were housed under the same conditions at the animal facilities of the French atomic energy commission (Commissariat à l'énergie atomique, CEA) in Grenoble (France) that has accreditation from the French authorities under the reference C38-18510001. Animal breeding, housing, and experiments were performed in strict accordance with the French animal testing regulation. The Atp7b -/-mice have been described earlier [START_REF] Burkhead | Systems biology approach to Wilson's disease[END_REF][START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF][START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF]; they were kindly given to us by Prof. Svetlana Lutsenko from John Hopkins University. Wild type (WT) specific-pathogen-free C57Bl/6 mice (Charles River Laboratories, Saint Germain Nuelles, France) were used as controls. The mice were kept on a 12h/12h light/dark cycle and fed with maintenance dry food #3469 (Kliba Nafag CH), containing 14 mg/kg copper, 60 mg/kg zinc and 250 mg/kg iron. Food and water were provided ad libitum.

Altogether, 137 Atp7b -/-(group Atp7b -/-) and 101 C57Bl/6 (group WT) mice were included in the study and separated into 4 groups according to age (6-8, 20, 39, and 50 weeks), chosen to illustrate different stages of WD [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF]. To provide insights into the effect of a Cu-chelator, 24 Atp7b -/-mice received D-penicillamine, from 39 to 50 weeks of age. Dpenicillamine was administered in the morning through a gastric tube, 5 days per week at a dose of 100 mg/kg body weight; a protocol that was approved by the French Research Ministry under the reference #2015062510212698_v1(APAFIS#926).

Animals were euthanized by CO2 inhalation at the indicated ages. Blood was immediately collected by cardiac exsanguination puncture either in sodium heparin tubes for enzyme measurements or in trace-element dedicated tubes (Becton Dickinson Vacutainer, #369032, Le Pont-de-Claix, France) for copper measurements. The serum was collected after coagulation and a 10-min centrifugation at 3000 rpm; it was kept at room temperature for further enzyme analysis. For copper evaluations, in each subgroup, total blood from different mice of the same sex were pooled up to ~4 mL, the volume required to perform all the copper

Analysis of aminotransferase activity and bilirubin

Serum total bilirubin, serum alanine (ALT), and aspartate (AST) aminotransferase activities were determined by automated measurement (Vista 1500, Siemens Health Care Diagnostics Inc., Newark, Delaware, US) performed routinely at the biochemistry laboratory of the Edouard Herriot Hospital. As these hepatic functions are considered not to vary over time in WT mice [START_REF] Schnell | Effect of blood collection technique in mice on clinical pathology parameters[END_REF], serum ALT, AST, and bilirubin were assessed only at 20 weeks of age, whereas they were evaluated for each Atp7b -/-subgroup.

Copper measurements in blood and liver

Total serum copper and intrahepatic copper were measured using an inductively coupled plasma optical emission spectrometer (ICP-OES Vistapro, Agilent Technologies, Santa Clara, California, US). For intrahepatic copper measurements, liver samples were dried overnight at 40°C then 3 hours at 105°C, followed by an acid digestion (HNO3 at 60°C overnight); results were expressed as µg of copper per gram of dry tissue.

CuEXC measurement was performed by Zeeman electrothermal atomic absorption spectrometry (ETAAS, ZAA220, Agilent Technologies, Santa Clara, California, US) according to a previously reported technique [START_REF] Balkhi | Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects[END_REF]. The lower limit of detection (LOD) was obtained over 30 determinations of the blank (NaCl 0.9%) and calculated as threefold the standard deviation (SD). The LOD was defined as 0.003 µmol/L. The precision (coefficient of Intraday precision ranged from 3.2 % to 5.1% for 21.7 µmol/L and 40 µmol/L of Cu in NaCl 0.9% respectively. REC was calculated by the ratio of CuEXC over total serum copper.

Histological analysis

Tissue sections were prepared by fixing livers in 10% buffered formalin at pH 7.4 for 3 days, transferring to 80% ethanol, and then embedding in paraffin. Each section was stained with hematoxylin eosin staining and fibrosis was investigated by Picro-Sirius red staining.

Histological analysis investigated and quantified fibrosis, inflammation, activity, cellular atypia, steatosis, nodules, and biliary abnormalities. Livers that were not analyzable (e.g. for reasons of poor fixing) were excluded; livers were analyzed at a 1:2 WT to Atp7b -/-ratio.

Statistical analysis

Quantitative variables were expressed as means (SD) and qualitative variables as frequencies or percentages. The main characteristics were compared between groups by using the Mann-Whitney test for continuous variables and the Chi-2 test for qualitative variables, with statistical significance set at a p value <0.05. Statistical analysis was performed with Sigma Plot 13 (Systat Software Inc.).

Results

Natural history and liver histology

A C C E P T E D M A N U S C R I P T
A total of 101 WT and 137 Atp7b -/-mice were included (Table 1); histological analysis was performed on a total of 192 livers from 64 WT and 128 Atp7b -/-mice, including 24 Atp7b - /-treated mice. No abnormality was noted in the WT group. Histological analysis of Atp7b -/- mice showed the progressive development of chronic liver injury with significant lesions, homogeneously distributed across each age group. At 6 weeks, no obvious macroscopic abnormality in color or shape was detected. Microscopic changes were absent or mild with a moderate inflammation (16/33=48%) and no significant fibrosis (Figure 1A andB). Over time, significant injuries appeared. At 50 weeks, lesions were extensive with constant intense inflammation, cellular atypia (e.g. swollen hepatocytes, nuclear enlargement, and atypical nucleoli; Figure 1C) and an extensive perisinusoidal fibrosis (Figure 1D). Nodules were noted in 16/25=65%. The use of copper chelator treatment significantly decreased injury compared to untreated mice at the same age (fibrosis: 66% vs. 100%; p<0.001). No malignant feature, dysplastic change, or significant biliary lesions were noted in any groups.

Hepatic functions

Hepatic functions were assessed at 20 weeks in WT mice (mean±SD, n=5: ALT 23±10 UI/L; AST 108±45 UI/L, and bilirubin <2 µmol/L), and were in agreement with previous data from C57Bl/6 mice [START_REF] Schnell | Effect of blood collection technique in mice on clinical pathology parameters[END_REF]. Serum ALT and AST regularly increased over time in the Atp7b -/- mice (n=5: from ALT 29±9 and AST 115±35 UI/L at 6-8 weeks to ALT 250±175 and AST 376±139 UI/L at 50 weeks, respectively). At 50 weeks, the mean levels of both aminotransferase activities were lowered by D-penicillamine (n=4: ALT 119±101 and AST 276±142) compared to those of untreated Atp7b -/-mice of the same age but the data scattering prevented any statistical significance. Bilirubin level increased in Atp7b -/-mice without reaching pathological values (<2 µmol/L at 6-8 weeks vs. 14.4±6.5 µmol/L at 50 weeks and 5.5±3.7 at 50 weeks + treatment).

A C C E P T E D

M A N U S C R I P T

Copper related markers

In WT mice, the mean ± SD Cus was 8.4±0.7 µmol/L at 6-8 weeks and 10.2±0.5 µmol/L at 50 weeks, IHCu concentrations was 14±4 µg/g dry liver at 6-8 weeks and 14±2 µg/g dry liver at 50 weeks, and CuEXC was 1.3±0.1 µmol/L at 6-8 weeks and 1.1±0.1 µmol/L at 50 weeks (Table 2). In Atp7b -/-mice, the mean ± SD Cus increased over 20 weeks and remained stable thereafter, IHCu was stable over time and CuEXC increased over 39 weeks and remained stable thereafter (Table 2 and Figure 2). In young mice (6-8 weeks), Cus was higher for the WT group than for the Atp7b -/-group (8.4±0.7 vs. 4.3±1.7 µmol/L, p<0.01), there was no difference at 20 weeks and Cus was significantly higher in Atp7b -/-mice after 39 weeks (Table 2). The IHCu concentration was stable over time in Atp7b -/-(Figure 2B) and always higher than in WT livers (Table 2). In young mice (6-8 weeks), CuEXC levels were similar in WT and Atp7b -/-mice and significantly higher in Atp7b -/-after 20 weeks (Table 2).

Treatment of Atp7b -/-mice with D-penicillamine led to significantly reduced mean±SD Cus concentration (9.6±2 vs. 13.3±1.2 µmol/L, p<0.01) and IHCu concentration (486±263 vs.

797±402 µg/g dry liver, p=0.026) and led to a non-significant trend towards decreased CuEXC (3.2±1.1 µmol/L vs. 4.6±2 µmol/L; p=0.07) at 50 weeks of age (Table 2). In the total population, the REC was significantly higher in Atp7b -/-compared to WT mice (39±12 vs. 11±3%, p<0.01; Figure 3A). The sex of animals (Figure 3B), the use of D-penicillamin (Figure 3C) and age (Figure 3D, Table 2), did not modify the REC value in Atp7b -/-mice. A cut-off value set at 20% conferred a 100% sensitivity and specificity, irrespective of age, sex, or use of treatment.

Discussion and conclusion

A C C E P T E D

M A N U S C R I P T
Since the discovery and cloning of the human ATP7B gene in 1993 [START_REF] Bull | The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene[END_REF][START_REF] Tanzi | The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene[END_REF], our knowledge of the genetic basis of WD has increased dramatically. However, the pathogenesis has not been fully deciphered and an explanation for the phenotypic variability is still missing.

Animal models of copper overload are therefore essential to understand the disease pathogenesis and improve the diagnosis and treatment of WD patients. Several rodent models have been described, for example, LEC rats and toxic milk mice are products of a spontaneous mutation in the Atp7b -/-gene that occurred during breeding [START_REF] Wu | The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene[END_REF][START_REF] Burkhead | Systems biology approach to Wilson's disease[END_REF][START_REF] Theophilos | The toxic milk mouse is a murine model of Wilson disease[END_REF].

However, the Atp7b -/-mouse, generated by Buiakova et al. [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF][START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF] is the only genetically engineered rodent strain that can serve as a model for the hepatic forms of WD. The insertion of multiple stop codons in exon 2 leads to truncated mRNA that are present in the tissues although the Atp7b protein is not produced [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF].

Although copper accumulation can be detected in other organs, the highest copper concentration and the most striking phenotype is observed in the livers of Atp7b -/-mice [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF].

Atp7b -/-mice are born with a copper deficiency but soon after copper accumulates in their liver and increases to peak levels between 6 and 20 weeks of age [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF][START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF]. In the present study, intrahepatic copper concentration was stable from 6 to 50 weeks of age and around 50fold the concentration of that found in WT mice. This copper overload led to hepatic tissue damage but there was no direct correlation between the IHCu concentration and the severity of the histological lesions as previously reported [START_REF] Huster | Structural and metabolic changes in Atp7b-/-mouse liver and potential for new interventions in Wilson's disease[END_REF]. One interpretation of these findings is that copper overload precedes tissue damage and that hepatic lesions occur not only because of copper toxicity per se but also through several mechanisms dealing with lipid metabolism, mitochondrial injury, and hepatic nuclear receptor dysfunction [START_REF] Huster | Structural and metabolic changes in Atp7b-/-mouse liver and potential for new interventions in Wilson's disease[END_REF][START_REF] Wooton-Kee | Elevated copper impairs hepatic nuclear receptor function in Wilson's disease[END_REF][START_REF] Huster | High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease[END_REF]. According to the literature, no acute liver failure was observed in Atp7b -/-mice; rather, they developed chronic hepatitis [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF]. Herein, numerous and systematic histological analyses of Atp7b -/-livers showed a progressive and homogenous development of chronic liver injury. At early stages of A C C E P T E D M A N U S C R I P T copper accumulation (6 weeks), no macroscopic morphological changes were observed in the liver and microscopic changes were moderate. Over time, serious injuries appeared leading to severe chronic liver disease in all mice at 50 weeks. At this stage, nodules were observed in two-thirds of the animals. This nodular evolution may be related to regenerative nodes, similar to those observed in human cirrhosis, or restoration of normal liver architecture [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF][START_REF] Huster | Structural and metabolic changes in Atp7b-/-mouse liver and potential for new interventions in Wilson's disease[END_REF]. Unlike previous descriptions [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF], we did not observe neoplastic biliary proliferation in older mice. The Golgi membrane protein GP73 has been recently identified as a potential trigger for fibrosis and dysplasia in Atp7b -/-mice [START_REF] Wright | Hepatocyte GP73 expression in Wilson disease[END_REF]. Further studies are needed to focus on this protein or other neoplastic processes. Some genetic drift may also have occurred since the creation of the original Atp7b -/-mice in 1999, leading to different phenotypes. For instance, Atp7b -/-mice born during the time of this study were nursed by their mother and none of them developed the neurological symptoms described earlier [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF]. Despite differences with previously published data, we observed very homogenous histological lesions in each Atp7b -/- subgroup leading to the hypothesis of a progressive worsening of hepatic lesion over time in Atp7b -/-mice. Herein, we confirm that the Atp7b -/-mouse model is a valuable model for chronic liver disease in WD and may be used to test new diagnostic tools or treatments.

CuEXC and REC have recently been described as new biomarkers for WD diagnosis in humans [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF]. In a previous report, El Balkhi et al. [START_REF] Balkhi | Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects[END_REF] described the ultrafiltration procedure and the instrumental determination of ultrafiltrable copper and CuEXC, and established reference values in healthy subjects. CuEXC and REC have appeared as highly sensitive and reliable tests for discriminating adults with WD from healthy subjects and also from heterozygous ATP7B mutation carriers [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF][START_REF] Trocello | Relative exchangeable copper: a promising tool for family screening in Wilson disease[END_REF]. The practicality of CuEXC determination may currently be a major limit of this new test. However, REC seems to represent a new specific, sensitive, and non-invasive biomarker for WD. Recently Guillaud et al. [START_REF] Guillaud 0 | Relative exchangeable copper: a valuable tool for the diagnosis of Wilson disease[END_REF] confirmed these data in humans, especially in the pediatric population and in adults or children with non-WD

A C C E P T E D M A N U S C R I P T hepatic diseases.
In this study a cut off value of 18.5% for the REC allowed to discriminate 49 WD patients from non-WD hepatic disease patients (103 adults and 49 children). Moreover, mean REC level was lower in cholestatic patients, in whom diagnosis is occasionally a problem, as increased IHCu may be observed. Further studies dealing with the measurement of the REC in other liver diseases would also be of great interest for humans and animals [START_REF] Fuentealba | Animal models of copper-associated liver disease[END_REF].

Recently, Schmitt et al. [START_REF] Schmitt | Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat[END_REF] have evaluated CuEXC and REC in LEC rats, an animal model of WD that mostly presents acute liver failure [START_REF] Kasai | Clinicopathological studies of LEC rats with hereditary hepatitis and hepatoma in the acute phase of hepatitis[END_REF][START_REF] Wu | The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene[END_REF]. The authors found that a cut off value for the REC set at 19% allowed to discriminate 24 LEC rats from 6 Long-Evans WT rats, with a sensitivity of 97.3% and a specificity of 100% [START_REF] Schmitt | Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat[END_REF]. In the present study, Atp7b -/-mice allowed us to include a greater number of cases but also to evaluate the evolution of copper parameters during chronic hepatitis as these do not present acute liver failure [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF]. Whereas recurrent episodes of acute liver failure can be observed over the course of a LEC rat's life, no mouse from the present study had such symptoms. Histological analysis found a progressive worsening of liver disease leading to fibrosis in all 50-week-old mice. We confirmed REC as a discriminative tool for the diagnosis of WD in mice; REC was significantly higher in Atp7b - /-mice than in WT. A cut off set at 20% gave sensitivity and specificity of REC of 100% in Atp7b -/-mice irrespective of sex, age, stage of the disease, or the use of a treatment.

Exchangeable copper (CuEXC) corresponds to the labile fraction of copper in the serum, not complexed to ceruloplasmin but complexed to albumin and other peptides [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF][START_REF] Balkhi | Determination of ultrafiltrable and exchangeable copper in plasma: stability and reference values in healthy subjects[END_REF].

An increase of CuEXC is thought to reflect a blood and tissue copper overload. If CuEXC (and its derived calculated REC) appears as an important new diagnostic biomarker in WD, it seems also that it could be used in monitoring the disease course and treatment efficacy.

Recently, a correlation was reported in LEC rats between CuEXC values and the severity of acute liver failure [START_REF] Schmitt | Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat[END_REF]; CuEXC was always lower in an animal receiving a low-copper diet.

Herein, no acute liver failure was observed but CuEXC tended to increase over time, in parallel with hepatic lesions. The sensitivity of LEC rats to dietary copper has been demonstrated [28]. The variation of CuEXC level according to the copper intake in LEC rats is consistent with the ability of CuEXC to reflect the copper overload [START_REF] Schmitt | Evolution of exchangeable copper and relative exchangeable copper through the course of Wilson's disease in the Long Evans Cinnamon rat[END_REF]. Recently Guillaud et al. [START_REF] Guillaud 0 | Relative exchangeable copper: a valuable tool for the diagnosis of Wilson disease[END_REF] showed a variability of CuEXC in human according to the observance of treatment in WD patients with significant higher CuEXC in non-compliant patients. In the present study, the use of D-Penicillamine reduced intrahepatic copper and significantly improved histological lesions. We observed a trend towards lower CuEXC in treated mice without reaching statistical difference. This result may be due to the reduced number of CuEXC values, but also to the small size of mice that does not allow measurement of CuEXC without sacrificing the animal. Moreover, we could not monitor the evolution of urinary copper concentration in treated mice to evaluate the efficacy of copper chelators for technical reasons. Additional studies are needed to assess the use of CuEXC as a monitoring marker of the disease course and of decoppering treatment efficacy in WD. Such studies should be performed on bigger mammals, if not on humans, to follow the disease course and its treatment on the same individual.

In conclusion, this study confirms REC as a discriminative tool for the diagnosis of WD in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment.

Illustrations

  disease (WD) is an autosomal recessive inherited disorder of copper metabolism due to loss-of-function mutations in the gene encoding the ATP7B protein, a Cu-transporting

  serum was then prepared and frozen at -80°C for 24-48h until sent for analysis in the Trace Element Laboratory of the Edouard Herriot Hospital, Lyon. Livers were removed rapidly after exsanguination and separated into 2 parts: the left lobe was fixed in formalin for histological analyses and the other lobes immediately frozen in liquid nitrogen and kept at -80°C.

  ) was determined for the two quality control (QC) levels, Normal range and High range Trace Element Serum Control (UTAK Laboratories Inc., Valencia, California, US). For the intraday assay, 30 replicates of each QC level were processed the same day.

  Siaj R, Sauer V, Stoppeler S, et al. Dietary copper triggers onset of fulminant hepatitis in the Long-Evans cinnamon rat model. World J Gastroenterol 2012; 18: 5542-50.

Figure 1 :

 1 Figure 1: Microscopic features illustrating the development of chronic liver injury over

Figure 2 :

 2 Figure 2: Evolution of copper related markers Atp7b -/-mice.

Figure 3 :

 3 Figure 3: Evolution of REC.

Table 1 : Atp7b -/-and WT mice groups according to age and sex.

 1

		U S C R I P T
		Group Atp7b -/-N	Group WT (C57Bl/6)
	Ages 6-8 weeks 20 weeks 39 weeks	male 16 12 15 M female 21 13 11 A total 37 25 26	male 16 3 0	female 16 26 20	Total 29 20 32
	50 weeks* 50 weeks + treatment All ages * five mice who were to be sacrificed at 50 weeks (without treatment) died before this time 17 8 25 0 20 20 12 12 24 72 65 137 19 82 101 point C C E P T E D
	A

Table 2 -Copper data from liver and serum Weeks IHCu a , µg/g dry liver Cus b , µmol/L CuEXC b , µmol/L REC b , %

 2 Mean values (±standard deviation, n), b Mean values (±standard deviation) n=5 for all serum data, * 0.01<p<0.05, ** p<0.01 for comparison WT vs Atp7b -/- at each age and with vs without treatment for the 50-week-old Atp7b -/-mice, ns p≥0.05 IHCu: Intrahepatic copper ; Cus: total serum copper; CuEXC: exchangeable copper ;REC: relative exchangeable copper.

		WT	Atp7b -/-	p	WT	Atp7b -/-	p	WT	Atp7b -/-	P	WT	Atp7b -/-	p
	6-8	14 (±4, 5) 617 (± 220, 16) **	8.4 (±0.7)	4.3 (±1.7)	** 1.3 (±0.1)	1.7 (±0.4) Ns	15.7 (±1)	43.9 (±4.3) **
	20	14 (±2, 5) 779 (±143, 9) **	11.0 (±0.9)	10.1 (±1.1)	ns 1.0 (± 0.2)	3.5 (±0.4) **	8.8 (±1.9)	35.2 (±6.6) **
	39	9 (±1, 5) 605 (±172, 16) **	7.7 (±0.2)	13.5 (±4.8)	*	0.8 (±0.0)	5.1 (±0.7) **	9.8 (±0.3)	42.1 (±16.1) **
	50	14 (±2, 5) 797 (±402, 9) **	10.2 (±0.5)	13.3 (±1.2)	** 1.1 (±0.1)	4.6 (± 2)	** 11.1 (±1.4) 34.9 (±15.4) **
	50 + treatment		486 (±263, 11) *		9.6 (±2)	**		3.2 (±1.1) Ns		33.9 (±9.9)	ns

a

Acknowledgment:

We thank personnel of Trace Element laboratory for his technical assistance. We thank P.

Robinson for reviewing this manuscript (Hospices Civils de Lyon).

We warmly acknowledge Pr. S. Lutsenko and Dr. D. Huster for kindly providing the Atp7b -/- mice and Dr. V. Brun, VetD, PhD for her kind help in organizing and performing the experiments on mice.

Financial support:

This work was supported by grants from the Agence Nationale pour la Recherche for AH and KU salaries (ANR-11-EMMA-025 "COPDETOX" and ANR-11-LABX-0003-01 LabEx ARCANE).