Vania Tacher
email: vania.tacher@aphp.fr

Islem Sifaoui

Rym Kharrat

Jean-Nicolas Nicolas Dacher

Virgile Chevance

Romain Gallet

Emmanuel Teiger

Hicham Kobeiter

Vincent Le Pennec

Alexis Jacquier

Haytham Derbel

Jean-François Deux

Vincent Lepennec

Damien Mandry

Laurent Macron

tomography angiography in the assessment of percutaneous left atrial appendage closure -Review and experts recommendations endorsed by the Société française d'imagerie cardiaque et vasculaire diagnostique et interventionnelle

Image analysis is divided into four steps that include analysis of the approach; search for a thrombus in the LAA; investigation of the anatomy of the LAA (morphology of the LAA, dimensions of the LAA and choice of device) and cardiac and thoracic assessments. Followup involves CT examination to check the position of the device and to detect any complications. On the basis of the results of currently available published research, a panel of experts has issued recommendations regarding cardiac CT angiography prior to percutaneous LAA closure device placement, which were further endorsed by the Société Française d'Imagerie Cardiaque et Vasculaire Diagnostique et Interventionnelle (SFICV).

List of abbreviations

Introduction

Atrial fibrillation (AF) is the most common arrhythmia and represents a public health issue with a projected incidence of up to 215,000 persons per year in Europe by 2030 [START_REF] Zoni-Berisso | Epidemiology of atrial fibrillation: European perspective[END_REF]. AF is common among the elderly and affects 30% of people over the age of 60 years [START_REF] Chugh | Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 Study[END_REF]. AF is responsible for over 15% of ischemic strokes and 25% of strokes between the ages of 80 and 89 years [START_REF] Wang | A risk score for predicting stroke or death in individuals with new-onset atrial fibrillation in the community: the Framingham Heart Study[END_REF]. In 90% of strokes, they are secondary to the migration of a thrombus from the left atrial appendage (LAA) whose formation was promoted by nonvalvular AF [START_REF] Wolf | Atrial fibrillation as an independent risk factor for stroke: the Framingham Study[END_REF].

Oral anticoagulants are the most effective prophylactic treatment of LAA thrombus [START_REF] Hart | Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibrillation[END_REF]; however they are generally underused due to the rigorous adherence required, their adverse effects and the narrowed therapeutic window [START_REF] Kirchhof | ESC Guidelines for the management of atrial fibrillation developed in collaboration with EACTS[END_REF]. Their administration is accompanied by a risk of hemorrhage and contraindications are common, especially in the elderly. Alternatives to oral anticoagulant therapy are surgical ligation of the LAA that significantly reduces the risk of stroke [START_REF] Blackshear | Appendage obliteration to reduce stroke in cardiac surgical patients with atrial fibrillation[END_REF], radiofrequency ablation of AF or, more recently, LAA percutaneous occlusion whose first implantation in humans was reported in 2001 [START_REF] Sievert | Percutaneous left atrial appendage transcatheter occlusion to prevent stroke in high-risk patients with atrial fibrillation: early clinical experience[END_REF][START_REF] Pacha | Percutaneous devices for left atrial appendage occlusion: A contemporary review[END_REF]. Two randomized trials have demonstrated the efficacy and safety of percutaneous LAA closure devices (LAAC) [START_REF] Reddy | 5-year outcomes after left atrial appendage closure: from the PREVAIL and PROTECT AF trials[END_REF][START_REF] Reddy | Percutaneous left atrial appendage closure vs warfarin for atrial fibrillation: a randomized clinical trial[END_REF][START_REF] Holmes | Prospective randomized evaluation of the Watchman left atrial appendage closure device in patients with atrial fibrillation versus long-term warfarin therapy: the PREVAIL trial[END_REF]. This document has been developed by ten experienced radiologists in cardiac imaging and two cardiologists involved in LAAC implantation. Based on the review of currently available published research and expert consensus, this document reflects the opinion of experts endorsed by the Société Française d'Imagerie Cardiaque et Vasculaire diagnostique et interventionnelle (SFICV) on imaging protocol, recommendations, a standardized analysis and a structured report involved in LAAC procedures for the radiologists.

Indication for percutaneous left atrial appendage occlusion

LAAC is considered in patients with nonvalvular AF with contraindication to anticoagulant therapy [START_REF] Holmes | Percutaneous closure of the left atrial appendage versus warfarin therapy for prevention of stroke in patients with atrial fibrillation: a randomised non-inferiority trial[END_REF]. In France, approximatively the number of persons eligible for percutaneous LAA occlusion is about 10, 000 to 30, 000 patients per year [START_REF] Cdlhad | Evaluation de l'occlusion de l'appendice auriculaire gauche par voie transcutanée[END_REF]. In 2020, the European Society of Cardiology recommendations have proposed LAAC as a therapeutic alternative in patients with AF and presenting a contraindication to anticoagulant therapy (Class IIb recommendation) [START_REF] Hindricks | ESC Guidelines for the diagnosis and management of atrial fibrillation developed in collaboration with the European Association for Cardio-Thoracic Surgery (EACTS)[END_REF]. The indication of LAAC is relevant in patients with AF with a definite and permanent contraindication to anticoagulants (validated by a multidisciplinary panel) and with high thromboembolic risk defined as a CHA2DS-VASc score  4 [START_REF] Has | Evaluation de l'occlusion de l'appendice auriculaire gauche par voie transcutanée[END_REF].

Technique

LAAC devices

Various models of percutaneous prostheses exist but two are predominantly used to exclude the LAA. They are the Watchman FLX TM device (Boston Scientific) and the Amplatzer TM Amulet TM device (Abbott Vascular) [START_REF] Staubach | Long-term transesophageal echocardiography follow-up after percutaneous left atrial appendage closure[END_REF][START_REF] Asmarats | Percutaneous left atrial appendage closure: current devices and clinical outcomes[END_REF].

The Watchman FLX TM device

The Watchman FLX TM device is the last generation of the Watchman device. The prosthesis consists of a cage-shaped nitinol frame structure partially covered with polyethylene terephthalate at its base and lateral edge. It rests against the internal face of the LAA (Supplemental Figure S1). Several observational studies and registries described the sizing measurements and the size of the device as well as the implantation modality of the device [START_REF] Masoudi | The evolution of left atrial appendage occlusion: EWOLUTION and the WATCHMAN in practice[END_REF][START_REF] Boersma | Implant success and safety of left atrial appendage closure with the WATCHMAN device: periprocedural outcomes from the EWOLUTION registry[END_REF][START_REF] Wang | Application of 3dimensional computed tomographic image guidance to WATCHMAN implantation and impact on early operator learning curve: single-center experience[END_REF][START_REF] Eng | Computed tomography for left atrial appendage occlusion case planning[END_REF][START_REF] Kaafarani | Role of CT imaging in left atrial appendage occlusion for the WATCHMAN device[END_REF].

The Amplatzer TM Amulet TM device

The self-expanding braided nitinol mesh prosthesis consists of an external disc resting against the atrial face of the LAA and an internal lobe positioned at the base of the LAA, with both parts linked by a central waist (Supplemental Figure S2). Characteristics of the device as well as its method of implantation have been described in several observational studies and registries [START_REF] Aminian | Incidence, characterization, and clinical Impact of device-related thrombus following left atrial appendage occlusion in the prospective global AMPLATZER amulet observational study[END_REF][START_REF] Landmesser | Left atrial appendage occlusion with the AMPLATZER Amulet device: one-year follow-up from the prospective global Amulet observational registry[END_REF][START_REF] Landmesser | Left atrial appendage occlusion with the AMPLATZER Amulet device: periprocedural and early clinical/echocardiographic data from a global prospective observational study[END_REF][START_REF] Clemente | Multimodality imaging in preoperative assessment of left atrial appendage transcatheter occlusion with the Amplatzer Cardiac Plug[END_REF].

Positioning the device

The pre-implantation (or pre-LAAC) assessment consists of a blood test (coagulation panel and serum creatinine value) and a cardiac computed tomography (CT) angiography [START_REF] Rajwani | CT sizing for left atrial appendage closure is associated with favorable outcomes for procedural safety[END_REF].

Transesophageal echocardiography (TEE) is almost always performed during system implantation. The procedure is performed under anesthesia (usually general) in an interventional imaging suite. An introducer sheath is placed in the right common femoral vein via femoral venous access. The device is contained in a support sheath mounted in the right atrium then introduced into the left atrium through an inter-atrial septum crossing. The device is then deployed under fluoroscopic and ultrasound guidance. In most cases the intervention takes place smoothly.

Pre-implantation imaging modalities

The imaging assessment includes cardiac CT angiography performed prior to implantation and TEE performed during the procedure [START_REF] Rajiah | Pre-and postprocedural CT of transcatheter left atrial appendage Closure devices[END_REF]. Although TEE has been considered as the gold standard, the three-dimensional nature of CT improves the accuracy for guiding the choice of LAAC device [START_REF] Chow | A comparative study of different imaging modalities for successful percutaneous left atrial appendage closure[END_REF][START_REF] Saw | Comparing measurements of CT angiography, TEE, and fluoroscopy of the left atrial appendage for percutaneous closure[END_REF][START_REF] Eng | Prospective, randomized comparison of 3-dimensional computed tomography guidance versus TEE data for left atrial appendage occlusion (PRO3DLAAO)[END_REF][START_REF] Morcos | Accuracy of commonly-used imaging modalities in assessing left atrial appendage for interventional closure: review article[END_REF]. It reduces the rate of peri-interventional complications compared to the TEE by improving the choice of prosthesis size. Investigating LAA with TEE rather than with CT might results in underestimation of the ostial dimensions and depth of the LAA [START_REF] Sievert | Percutaneous left atrial appendage transcatheter occlusion to prevent stroke in high-risk patients with atrial fibrillation: early clinical experience[END_REF].

Lastly, CT enables vascular approach, transeptal puncture site and circumflex artery location to be anticipated prior to implantation [START_REF] Ismail | CT imaging for left atrial appendage closure: a review and pictorial essay[END_REF].

CT image acquisition protocol prior to LAAC

The purpose of pre-implantation cardiac CT angiography is to evaluate the anatomy of the LAA in order to select the most suitable prosthesis and check for any contraindications to introducing the device. Patients should be preferably in a nonfasting state. Imaging has been recommended to be performed using CT unit with at least 64 rows of detectors, with electrocardiogram (ECG)-gating [START_REF] Korsholm | Cardiac Computed Tomography for Left atrial appendage Occlusion: acquisition, analysis, advantages, and Limitations[END_REF]. Both prospective and retrospective ECG-gating have been used for all pre-procedural imaging acquisitions [START_REF] Korsholm | Cardiac Computed Tomography for Left atrial appendage Occlusion: acquisition, analysis, advantages, and Limitations[END_REF]. Prospective ECG gating is more sensitive to cardiac arrhythmia and does not allow post hoc reconstruction of cardiac phases, but the radiation exposure is significantly reduced compared with retrospective ECG gating.

Images should be obtained in a phase corresponding to 30-60% of the R-R interval to reduce radiation exposure [START_REF] Patel | Cardiac cycledependent left atrial dynamics: implications for catheter ablation of atrial fibrillation[END_REF]. Based on currently available published research and experts consensus, we recommend a prospective triphasic ECG-gated acquisitions with a spontaneous contrast phase followed with an arterial and a venous phases (70 s post-injection) with 1 mL of iodinated contrast material per kg (concentration ≥ 350 mg iodine/mL) at a rate of 4-5 mL/s flushed with 50 mL of saline solution [START_REF] Korsholm | Expert recommendations on cardiac computed tomography for planning transcatheter left atrial appendage occlusion[END_REF]. The non-enhanced phase may be considered for adjustment of the field of view before contrast media injection [START_REF] Korsholm | Expert recommendations on cardiac computed tomography for planning transcatheter left atrial appendage occlusion[END_REF][START_REF] Bilchick | Effectiveness of integrating delayed computed tomography angiography imaging for left atrial appendage thrombus exclusion into the care of patients undergoing ablation of atrial fibrillation[END_REF]. Acquisition during the venous phase is useful if filling of the LAA for the arterial phase is incomplete (a frequent situation) and if the presence of a thrombus in the LAA cannot be ruled out. Dualenergy CT is also valuable for differentiating between delayed opacification and a thrombus [START_REF] Hur | Cardioembolic stroke: dual-energy cardiac CT for differentiation of left atrial appendage thrombus and circulatory stasis[END_REF].

All these parameters are noted in the suggested standardized report detailed in Table 1. In case of contraindication of iodinated contrast media injection, cardiac magnetic resonance imaging is a valuable tool for detecting intra-LAA thrombus [START_REF] Chen | Cardiac MRI for detecting left atrial/left atrial appendage thrombus in patients with atrial fibrillation: meta-analysis and systematic review[END_REF].

Image analysis

For consistency in image analysis and report, we recommend four steps in image analysis as in the standardized report (Table 1): analysis of the approach, search for a thrombus in the LAA, investigating LAA anatomy, and cardiac and thoracic assessments.

Analysis of the approach

Analysis of the approach includes examining the inferior vena cava included in the field of view and the right atrium, through which the device passes. The interatrial septum (IAS) should also be analyzed for abnormality (interatrial communication, mass, thrombus, etc.) or anatomical variation (interatrial septal aneurysm, patent foramen ovale, lipomatous hypertrophy of the interatrial septum that may interfere with the transseptal puncture (Supplemental Figure S3). The IAS analysis provides optimal transseptal puncture position choice to allow device and LAA alignment.

Search for a thrombus in the LAA

The presence of a thrombus in the LAA is a contraindication for device placement. It manifests as a hypo attenuating structure partially or totally occupying in the LAA, visible on the arterial phase acquisition and persisting on the venous phase acquisition (Supplemental Figure S4). It is important to differentiate between delayed filling of the LAA and a thrombus.

A delay in opacification presents as an LAA opacification defect that disappears in the venous phase. This is almost always due to a large volume left atrium circulating slowly. The ratio of iodinated contrast material attenuation/native blood attenuation is indicative of stasis, not thrombus but the ratio cut-offs vary substantially between publications, making clinical application using the ratio alone difficult [START_REF] Kim | Differentiation between spontaneous echocardiographic contrast and left atrial appendage thrombus in patients with suspected embolic stroke using two-phase multidetector computed tomography[END_REF][START_REF] Spagnolo | Diagnosis of left atrial appendage thrombus in patients with atrial fibrillation: delayed contrast-enhanced cardiac CT[END_REF][START_REF] Romero | Cardiac imaging for assessment of left atrial appendage stasis and thrombosis[END_REF]. When two acquisitions (arterial and venous) are performed, the sensitivity, specificity and negative predictive value of CT for detecting a thrombus in the LAA are 97%, 100% and 97%, respectively [START_REF] Romero | Cardiac imaging for assessment of left atrial appendage stasis and thrombosis[END_REF][START_REF] Romero | Detection of left atrial appendage thrombus by cardiac computed tomography in patients with atrial fibrillation: a meta-analysis[END_REF].

Investigating the LAA anatomy

This step analyzes the overall morphology of the LAA, its dimensions and its relationship to the left circumflex artery that is potential risk of compression by the device.

The analysis is preferably performed in the ventricular systolic phase to ensure maximum atrial filling. The LAA appears on the scan as a blind structure, often pyramidal in shape, with extensions known as lobes [START_REF] Naksuk | Left atrial appendage: embryology, anatomy, physiology, arrhythmia and therapeutic intervention[END_REF]. Its anatomy and dimensions vary widely among individuals, without any correlation with patient size and gender [START_REF] Veinot | Anatomy of the normal left atrial appendage: a quantitative study of age-related changes in 500 autopsy hearts: implications for echocardiographic examination[END_REF]. The CT report must specify:

1. LAA morphology

LAA morphology can be seen on multiplanar reconstruction and three-dimensional volume reconstructions. Wang et al. described 4 different morphologies: the "chicken wing" type (Supplemental Figure S5), the most common type (48% of patients), the "cactus" type (30% of patients) (Supplemental Figure S6); the "windsock" type (19% of patients) (Supplemental Figure S7) and the "cauliflower" type (3% of patients) (Supplemental Figure S8) [START_REF] Wang | Application of 3dimensional computed tomographic image guidance to WATCHMAN implantation and impact on early operator learning curve: single-center experience[END_REF]. The "cauliflower" type is associated with a higher embolic risk while the "chicken wing" type represents the least risk [START_REF] Wang | Left atrial appendage studied by computed tomography to help planning for appendage closure device placement[END_REF][START_REF] Lupercio | Left atrial appendage morphology assessment for risk stratification of embolic stroke in patients with atrial fibrillation: a meta-analysis[END_REF][START_REF] Romero | Left atrial appendage morphology and physiology: "the missing piece in the puzzle[END_REF]. The latter also seems to be the most problematic for LAAC device implantation because of its sharp angulation [START_REF] Biase | Does the left atrial appendage morphology correlate with the risk of stroke in patients with atrial fibrillation? Results from a multicenter study[END_REF]. The number of lobes of the LAA must also be specified as the presence of several lobes may compromise the device's seal.

There are usually two lobes (54% of patients) (Supplemental Figure S9). More rarely, one lobe (20% of patients), 3 lobes (25% of patients) and rarely 4 lobes (< 10% of patients) are found [START_REF] Veinot | Anatomy of the normal left atrial appendage: a quantitative study of age-related changes in 500 autopsy hearts: implications for echocardiographic examination[END_REF]. The presence of a lobe emerging from the base of the LAA must also be reported as this may lead to a leak in the LAA closure device. Such lobe has to be distinguished from accessory appendage [START_REF] Abbara | Cardiac CT assessment of left atrial accessory appendages and diverticula[END_REF].

LAA dimensions

Each device manufacturer provides its own guidelines for determining the sizing of device to implant [START_REF] Landmesser | Left atrial appendage occlusion with the AMPLATZER Amulet device: one-year follow-up from the prospective global Amulet observational registry[END_REF][START_REF] Landmesser | Left atrial appendage occlusion with the AMPLATZER Amulet device: periprocedural and early clinical/echocardiographic data from a global prospective observational study[END_REF][START_REF] Xu | Computed tomography measurement of the left atrial appendage for optimal sizing of the Watchman device[END_REF][START_REF] Tzikas | Left atrial appendage occlusion with the AMPLATZER Amulet device: an expert consensus stepby-step approach[END_REF]. The LAA is measured in double oblique view to distinguish a slice passing through the LAA, the left upper pulmonary vein and the circumflex artery (Figure 1) [START_REF] Korsholm | Transcatheter left atrial appendage occlusion in patients with atrial fibrillation and a high bleeding risk using aspirin alone for post-implant antithrombotic therapy[END_REF]. Kirchhof et al. suggest obtaining this slice by successively applying a right anterior obliquity of 30° on the axial slice and a cranial obliquity of 10° on the sagittal plane [START_REF] Kirchhof | ESC Guidelines for the management of atrial fibrillation developed in collaboration with EACTS[END_REF]. The pre-implantation CT measurements to be taken for the 2 types of device are: (i), measurement of the anatomical orifice (AO): This is located at the junction between the LAA and the right atrium. The large and small diameters must be measured (Figure 2); and (ii), measurement of the implantation zone (IZ) (or "landing zone"): This is located at the entrance of the main lobe where the device provides a perfect seal because it is confined by the LAA walls. It is usually measured at approximately 10 mm from the AO. The large and small diameters must be noted (Figure 3). Additional measurements specific to each device are also required. For the Watchman system this is the maximum depth of the LAA corresponding to the distance between the center of the anatomical orifice and the apex of the main lobe, the latter being unfolded over a double oblique slice (Figure 4). For the Amplatzer Amulet system, the direct depth must be measured. This is the orthogonal distance between the center of the anatomical orifice and the adjacent base of the LAA (Figure 5). Figure 6 summarizes all the dimensions to be recorded according to the device type.

Selection of device

Measurements on CT images depend on the type of prosthesis. For the Watchman device, the size of the device is selected by oversizing the large diameter of the landing zone of the LAA by 10 to 30 % using a specific table available on the manufacturer's site. There are five different sizes ranging from 20-to 35 mm in diameter. The length of the device must be less than the maximum depth of the LAA. For the Amulet, the largest diameter of the landing zone is oversized by 3-to 6 mm. The depth of the device must be at least 2.5 mm less than the depth of the LAA. There are 8 different diameter sizes ranging from 16 to 34 mm.

Cardiac and thoracic assessments

In addition to AAG related procedure assessment, CT helps perform coronary calcium score quantification with the first enhanced ECG-gated examination and eventually add coronary heart disease risk estimation in those patients. Coronary analysis may be performed when image quality allows its analysis because CT image quality may be impaired in patients with AF [START_REF] Katona | Left circumflex coronary artery occlusion due to a left atrial appendage closure device[END_REF]. This information may modify patients care management [START_REF] Alkindi | Anticoagulation in patients with atrial fibrillation and coronary artery disease[END_REF][START_REF] Knuuti | ESC Guidelines for the diagnosis and management of chronic coronary syndromes[END_REF]. CT allows the assessment of the entire cardiac mass and all adjacent structures such as mediastinum, vascular anatomy, pulmonary parenchyma, upper abdominal slices, bone structures to search for incidental abnormalities [START_REF] Cellina | CT features of an uncommon association between coronary-pulmonary fistula and Vieussens' arterial ring[END_REF][START_REF] Raimondi | Imaging features of complete congenital atresia of left coronary artery[END_REF].

Peri-procedural LAAC device related complications

Per-intervention complications have been reported such as perforation of the LAA responsible for hemopericardium with tamponade (if the device is oversized), migration of the device (if the device is undersized), that could be immediately been detected with TEE. When suspected, early complications such as compression of the circumflex artery by the device responsible for myocardial ischemia, gas embolism, infection and hematoma at the puncture point can be depicted with CT [START_REF] Rajwani | CT sizing for left atrial appendage closure is associated with favorable outcomes for procedural safety[END_REF][START_REF] Katona | Left circumflex coronary artery occlusion due to a left atrial appendage closure device[END_REF]. Other complication as stroke/transient ischemic attack are explored with CT and /or magnetic resonance imaging. Late complications such as migration of the device, major or minor peri-prosthetic leak or atrial thrombus are better depicted with CT than with TEE [START_REF] Masoud | Outcome of left atrial appendage occlusion in high-risk patients[END_REF].

CT-related radiation exposure

Mean radiation with standard cardiac protocol is less than 3 mSv [START_REF] Spagnolo | Diagnosis of left atrial appendage thrombus in patients with atrial fibrillation: delayed contrast-enhanced cardiac CT[END_REF][START_REF] Saw | Cardiac CT angiography for device surveillance after endovascular left atrial appendage closure[END_REF][START_REF] Gerber | Radiation dose and safety in cardiac computed tomography[END_REF]. The advent of low-dose acquisition as well as hybrid reconstruction may lower the radiation dose delivered to the patient [START_REF] Beregi | Low and ultra-low dose radiation in CT: opportunities and limitations[END_REF][START_REF] Greffier | CT dose optimization for the detection of pulmonary arteriovenous malformation (PAVM): a phantom study[END_REF].

Follow-up

No consensus or guidelines are available regarding the imaging follow-up after LAAC procedure (modality and frequency). Routinely, imaging follow-up is performed by TEE at 24-48 h to identify any pericardial effusion that may suggest rupture or migration of the system. After the hospital stay, follow-up involves regular scanning to check the correct position of the device and detect any migration (Supplemental Figure S10) [START_REF] Saw | Cardiac CT angiography for device surveillance after endovascular left atrial appendage closure[END_REF][START_REF] Poulter | Cardiac computed tomography follow-up of left atrial appendage exclusion using the Amplatzer Cardiac Plug device[END_REF][START_REF] Lakkireddy | Rationale and design for AMPLATZER amulet left atrial appendage Occluder IDE randomized controlled trial (Amulet IDE Trial)[END_REF]. The formation of a thrombus in contact with the atrial face of the system (Supplemental Figure S11), a rare but potentially serious complication, should also be recorded [START_REF] Saw | Cardiac CT angiography for device surveillance after endovascular left atrial appendage closure[END_REF]. Its presence imposes that anticoagulant treatment must be introduced. CT-scanner should also look for residual opacification of the LA (Supplemental Figure S12), which is found in more than 50% of patients, reflecting a system leak whose clinical impact remains controversial [START_REF] Saw | Incidence and clinical impact of device-associated thrombus and peri-device leak following left atrial appendage closure with the Amplatzer cardiac plug[END_REF].

Complete or partial, it may be visible from the arterial phase or only in the venous phase [START_REF] Hornung | Catheter-based closure of residual leaks after percutaneous occlusion of the left atrial appendage[END_REF].

It should be noted that CT appears more sensitive than TEE in detecting leaks after device implantation [START_REF] Jaguszewski | Cardiac CT and echocardiographic evaluation of peri-device flow after percutaneous left atrial appendage closure using the AMPLATZER cardiac plug device[END_REF][START_REF] Lim | Delayed left atrial appendage contrast filling in computed tomograms after percutaneous left atrial appendage occlusion[END_REF].

Figure legends

AF:

  Atrial fibrillation; AO: Anatomical orifice; CT: Computed tomography; ECG: Electrocardiogram; IZ: Implantation zone (or "landing zone"); LAA: Left atrial appendage; LAAC: LAA closure; SFICV: Société Française d'Imagerie Cardiaque et Vasculaire diagnostique et interventionnelle; TEE: Transesophageal echocardiography Mesh terms (There are five index terms)

Figure 1 :

 1 Figure 1:Reference arterial phase ECG-gated CT image in the sagittal oblique plane that allows analysis of the left atrial appendage in a patient with atrial fibrillation candidate for a left atrial appendage closure. The first step consists of showing the base, the main lobe, and the tip of the left atrial appendage (star) on a double oblique incidence including the circumflex artery (arrowhead) and the left upper pulmonary vein (arrow). A stent is present in the circumflex artery, resulting in few metal artifacts.

Figure 2 :

 2 Figure2: Arterial phase reference ECG-gated CT images allowing analysis of the anatomical orifice of the left atrial appendage. A, On the reference CT image in the sagittal oblique plane, the anatomical orifice of the left atrial appendage is represented with a red line. B, On CT image obtained in the orthogonal plane relative to A, the anatomical orifice is measured. The red lines represent the measurements of the large and small diameters of the anatomical orifice zone.

Figure 3 :

 3 Figure 3:Reference arterial phase ECG-gated CT images allowing analysis of the left atrial appendage landing zone. A, On the reference CT image in the sagittal oblique plane, the left atrial landing zone is shown here as a red line, located 10 mm above the anatomical orifice. B, On CT image obtained in the orthogonal plane relative to A, the landing zone is measured. The red lines represent the measurements of the large and small diameters of the landing zone.

Figure 4 :

 4 Figure 4:Reference arterial phase ECG-gated CT image in the sagittal oblique plane allowing analysis of the maximum depth of the left atrial appendage. On the reference slice, the thick white line represents the measurement of the maximum depth between the center of the anatomical orifice (thin white line) and the base of the main lobe of the left atrial appendage.

Figure 5 :

 5 Figure 5:Reference arterial phase ECG-gated CT image in the sagittal oblique plane allowing analysis of the direct depth of the left atrial appendage. The fine white line represents the measurement of the direct perpendicular depth between the center of the anatomical orifice (thick white line) and the edge of the main lobe of the left atrial appendage.

Figure 6 :

 6 Figure6: Diagram summarizing the measurements to be taken from the reference slice before implantation of a left atrial appendage closure device: the anatomical zone, the landing zone, the direct depth and the maximum depth.

Table 1 :

 1 Standardized CT report before left atrial appendage closure.

Table 2 :

 2 Recommendations for CT work-up in patients candidate for LAAC device placement and for post LAAC device placement follow-up.

Funding

This work did not receive any grant from funding agencies in the public, commercial, or notfor-profit sectors.

Conclusion

This review with recommendations (Table 2) and a standardized report is a support for the clinician workflow for pre-planning and follow-up cardiac computed tomography angiography for LAAC procedure.

Human rights

The authors declare that the work described has been carried out in accordance with the Declaration of Helsinki of the World Medical Association revised in 2013 for experiments involving humans.

Informed consent and patient details

The authors declare that this report does not contain any personal information that could lead to the identification of the patients.

Disclosure of interest

The authors declare that they have no competing interest.

Author contributions

All authors attest that they meet the current International Committee of Medical Journal Editors (ICMJE) criteria for Authorship.


Anne Platel
email: anne.platel@pasteur-lille.fr

Romain Dusautoir
email: dusautoir.romain@gmail.com

Gwenola Kervoaze
email: gwenola.kervoaze@pasteur-lille.fr

Gonzague Dourdin
email: gonzague.dourdin@pasteur-lille.fr

Eulalie Gateau
email: eulalie.gateau@pasteur-lille.fr

Smail Talahari
email: smail.talahari@pasteur-lille.fr

Ludovic Huot
email: ludovic.huot@pasteur-lille.fr

Sophie Simar
email: sophie.simar@pasteur-lille.fr

Anais Ollivier
email: anais.ollivier@pasteur-lille.fr

William Laine
email: william.laine@univ-lille.fr

Smaïl Talahari

Anaïs Ollivier

Jérôme Kluza
email: jerome.kluza@inserm.fr

Philippe Gosset
email: philippe.gosset@pasteur-lille.fr

Guillaume Garçon
email: guillaume.garcon@univ-lille.fr

Sébastien Anthérieu
email: sebastien.antherieu@univ-lille.fr

Jean-Marc Lo Guidice
email: jean-marc.lo-guidice@univ-lille.fr

Fabrice Nesslany
email: fabrice.nesslany@pasteur-lille.fr

Jean-Marc Lo

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures

Keywords: micronucleated polychromatic erythrocytes, PCE: polychromatic erythrocytes, NCE: normochromatic erythrocytes, RET: reticulocytes, RBC: red blood cells, PAHs: polycyclic aromatic hydrocarbons, ALI: air-liquid interface

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Tobacco use is a major public health concern killing more than 8 million people every year worldwide, yet is the leading cause of preventable death worldwide (World Health Organization, 2020).

Cigarette smoke is composed of a mixture of toxicants and carcinogens such as polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, aromatic amines, aldehydes (e.g. formaldehyde and acetaldehyde), phenols, volatile hydrocarbons, and metals (International Agency for Research on Cancer, 2012Cancer, , 2004)). Tobacco smoking is classified as a human carcinogen (group 1) by the International Agency for Research on Cancer (IARC) for the development of mainly lung cancer and several other cancers (larynx, pharynx, oesophagus, stomach, colon, liver, pancreas, bladder, cervix, ...) (International Agency for Research on Cancer, 2012Cancer, , 2004)). It is also well known that smoking is a major risk factor for many other adverse effects on human health, including respiratory, cardiovascular, nervous, immune, liver, urinary, gastrointestinal and reproductive systems [START_REF] Altamirano | Cigarette smoking and chronic liver diseases[END_REF][START_REF] Dechanet | Effects of cigarette smoking on reproduction[END_REF][START_REF] Erhardt | Cigarette smoking: An undertreated risk factor for cardiovascular disease[END_REF][START_REF] Gotts | What are the respiratory effects of e-cigarettes?[END_REF][START_REF] Lakier | Smoking and cardiovascular disease[END_REF][START_REF] Li | Cigarette smoking and gastrointestinal diseases: The causal relationship and underlying molecular mechanisms (Review)[END_REF][START_REF] Orth | Smoking -A Renal Risk Factor[END_REF][START_REF] Soares | Cigarette smoking and reproductive function[END_REF][START_REF] Sopori | Effects of cigarette smoke on the immune system[END_REF][START_REF] Sopori | Immunomodulatory effects of cigarette smoke[END_REF]. In recent years, a wide variety of new products, in particular electronic cigarettes (e-cigs), have emerged on the market as an alternative to smoking tobacco products. E-cigs are battery-powered devices that allow the nebulization of e-liquids composed of propylene glycol and/or glycerol and flavoring agents, with or without nicotine. E-cigs are generally perceived as less harmful than traditional cigarettes, particularly because they do not contain tobacco, do not require combustion during use and deliver fewer toxicants [START_REF] Cao | Toxicity of electronic cigarettes: A general review of the origins, health hazards, and toxicity mechanisms[END_REF][START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF]. However, due to the thermal degradation of the e-liquid constituents, other substances in e-cig aerosols have been identified as toxic compounds or potential carcinogens such as aldehydes (e.g. formaldehyde, acetaldehyde, methylglyoxal, acrolein), phenolic compounds (e.g. phenol, quinol, catechol, ortho-, meta-and para-cresol), volatile organic compounds (e.g. xylene, toluene, acetonitrile) and heavy metals (e.g. nickel and copper) [START_REF] Beauval | Influence of puffing conditions on the carbonyl composition of ecigarette aerosols[END_REF][START_REF] Beauval | Chemical Evaluation of Electronic Cigarettes: Multicomponent Analysis of Liquid Refills and their Corresponding Aerosols[END_REF][START_REF] Beauval | Trace elements in e-liquids -Development and validation of an ICP-MS method for the analysis of electronic cigarette refills[END_REF][START_REF] Cao | Toxicity of electronic cigarettes: A general review of the origins, health hazards, and toxicity mechanisms[END_REF][START_REF] Erythropel | Formation of flavorant-propylene Glycol Adducts With Novel Toxicological Properties in Chemically Unstable E-Cigarette Liquids[END_REF][START_REF] Gillman | Effect of variable power levels on the yield of total aerosol mass and formation of aldehydes in e-cigarette aerosols[END_REF][START_REF] Merecz-Sadowska | A Summary of In vitro and In vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment[END_REF][START_REF] Polosa | The effect of e-cigarette aerosol emissions on respiratory health: a narrative review[END_REF]. Moreover, due to the lack of in-depth toxicity assessment, especially long term or repeated-dose toxicity studies, safety of e-cigs cannot be guaranteed.

Recent reviews [START_REF] Cao | Toxicity of electronic cigarettes: A general review of the origins, health hazards, and toxicity mechanisms[END_REF][START_REF] Merecz-Sadowska | A Summary of In vitro and In vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment[END_REF][START_REF] Polosa | The effect of e-cigarette aerosol emissions on respiratory health: a narrative review[END_REF][START_REF] Wang | Toxicity assessment of electronic cigarettes[END_REF] provide an overview of the in vitro and in vivo toxicity of e-cigs. To date, the published studies mainly focus on in vitro toxic effects of e-cigs emissions. Data show that exposure to e-cig aerosols triggers cytotoxic effects such as cell death, DNA damage, and reactive oxygen species (ROS) and proinflammatory agents' production. It was also demonstrated that e-cig aerosol is much less cytotoxic than traditional cigarette smoke [START_REF] Anthérieu | Comparison of cellular and transcriptomic effects between electronic cigarette vapor and cigarette smoke in human bronchial epithelial cells[END_REF][START_REF] Cervellati | Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells[END_REF][START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF][START_REF] Neilson | Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol[END_REF][START_REF] Tellez | Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines[END_REF]. In vitro, e-cigs had the potential to increase oxidative stress and inflammatory response in a similar level to that of cigarette smoke, but after more intensive exposures [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF]. In contrast, there are relatively few in vivo experimental approaches. Most of them were performed or sponsored by the tobacco industry and have been carried out using short-term exposures (from a few hours to a few weeks), with individual e-cig components (e.g. popylene glycol and/or vegetable glycerine) but not whole aerosols, or with old generation or low-power e-cig devices.

Furthermore, the experimental protocols of exposure often used did not correspond to normal conditions of use (e.g. number of puffs/min), or used whole body exposure systems that are not representative of real exposure since animals are exposed by other routes than the respiratory route (i.e. cutaneous and digestive by deposition of e-cig aerosols on the coat). Another limitation of most of these studies is that they did not compare the results obtained with e-cig aerosol with those obtained with traditional cigarette smoke. Results showed that e-cig aerosols are likely to induce oxidative stress, mitochondrial dysfunction, pulmonary inflammation, DNA damage and even impairment of respiratory function [START_REF] Canistro | E-cigarettes induce toxicological effects that can raise the cancer risk[END_REF][START_REF] Garcia-Arcos | Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner[END_REF][START_REF] Glynos | Comparison of the effects of e-cigarette vapor with cigarette smoke on lung function and inflammation in mice[END_REF][START_REF] Hwang | Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria[END_REF][START_REF] Laube | Acute and chronic in vivo effects of exposure to nicotine and propylene glycol from an Ecigarette on mucociliary clearance in a murine model[END_REF][START_REF] Lerner | Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts[END_REF][START_REF] Lim | Inhallation of e-Cigarette Cartridge Solution Aggravates Allergen-induced Airway Inflammation and Hyper-responsiveness in Mice[END_REF][START_REF] Mcgrath-Morrow | The Effects of Electronic Cigarette Emissions on Systemic Cotinine Levels, Weight and Postnatal Lung Growth in Neonatal Mice[END_REF][START_REF] Salturk | Effects of Electronic Nicotine Delivery System on Larynx: Experimental Study[END_REF][START_REF] Scott | Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages[END_REF][START_REF] Werley | Toxicological assessment of a prototype e-cigaret device and three flavor formulations: a 90-day inhalation study in rats[END_REF].

Regarding the assessment of the genotoxicity of tobacco products, many studies have been conducted to specifically investigate the in vitro genotoxic/mutagenic potential of cigarette smoke and ecig aerosols. Unfortunately, contradictory results have often been obtained depending on the products tested (e.g. particulate phase, gas phase, smoke condensate or extract, e-liquid itself, or whole smoke aerosol), the cell line used (e.g. lung epithelial cells, oral epithelial cells, oropharynx cells, or 'regulatory' cells), or the mode of cell exposure [(e.g. submerged cell cultures or air-liquid interface (ALI) conditions)].

For these reasons, some of these cell treatment/exposure methods are not representative of an actual human exposure to cigarette smoke or e-cig aerosols. In the Ames test, negative responses were observed with e-liquid and e-cig aerosols (with nicotine and a range of flavorings) whereas positive results were obtained concurrently with 3R4F smoke [START_REF] Wieczorek | A comparative in vitro toxicity assessment of electronic vaping product eliquids and aerosols with tobacco cigarette smoke[END_REF]. Similar negative results were obtained with e-liquids and pad-collected aerosols of e-cigs, and positive results with pad-collected smoke condensates of tobacco cigarettes (3R4F, 1R5F, Malboro gold) [START_REF] Misra | Comparative In vitro Toxicity Profile of Electronic and Tobacco Cigarettes, Smokeless Tobacco and Nicotine Replacement Therapy Products: E-Liquids, Extracts and Collected Aerosols[END_REF]. Rudd et al. also demonstrated that e-cig emission is not mutagenic under their tested conditions, unlike 3R4F cigarette smoke [START_REF] Rudd | Chemical Composition and In vitro Toxicity Profile of a Pod-Based E-Cigarette Aerosol Compared to Cigarette Smoke[END_REF]. Thorne et al. have carried out a study on Salmonella typhimurium strains TA98 and TA100 exposed at the air-agar interface to e-cig aerosols and showed no mutagenic activity in contrast to 3R4F cigarette smoke [START_REF] Thorne | The mutagenic assessment of an electronic-cigarette and reference cigarette smoke using the Ames assay in strains TA98 and TA100[END_REF]. The same authors also performed a mouse lymphoma assay at the tk locus and in vitro micronucleus tests (on CHO, V79 and TK6 cells) with an eliquid, the e-cig aerosol matter captured from the same e-liquid, and the total particulate matter from a 3R4F cigarette. No mutagenic or genotoxic effect was observed for the e-liquid and its aerosol, in contrast to 3R4F smoke (Thorne et al., 2019a(Thorne et al., , 2019b)). All in vitro micronucleus tests reported in the literature were negative [START_REF] Misra | Comparative In vitro Toxicity Profile of Electronic and Tobacco Cigarettes, Smokeless Tobacco and Nicotine Replacement Therapy Products: E-Liquids, Extracts and Collected Aerosols[END_REF][START_REF] Rudd | Chemical Composition and In vitro Toxicity Profile of a Pod-Based E-Cigarette Aerosol Compared to Cigarette Smoke[END_REF][START_REF] Tellez | Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines[END_REF]Thorne et al., 2019a;[START_REF] Wieczorek | A comparative in vitro toxicity assessment of electronic vaping product eliquids and aerosols with tobacco cigarette smoke[END_REF], either with e-liquids, aerosols or condensates, except the one reported very recently by [START_REF] Tellez | Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines[END_REF] on e-cig aerosols (containing diverse flavoring product, with and without nicotine) in oral epithelial cells. In contrast, in all these studies, traditional cigarette smoke (or total particulate matter or condensate) induced chromosomal aberrations. Very recently, Tellez et al. demonstrated that 10 different e-cig aerosols did not induce DNA damage, as measured by the in vitro comet assay, in oral epithelial cells, unlike the 3R4F cigarette [START_REF] Tellez | Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines[END_REF]. This result was not confirmed by several previously published data. Khalil et al., also using the in vitro comet assay, showed that e-cig aerosols cause DNA damage in A549 lung cells exposed at the ALI [START_REF] Khalil | E-cigarette aerosol induced cytotoxicity, DNA damages and late apoptosis in dynamically exposed A549 cells[END_REF]. Ganapathy et al. also reported that ecig aerosol extracts can induce significant increases in DNA damage (using the primer anchored DNA damage detection assay), including 8-OHdG, on human oral and lung epithelial cells [START_REF] Ganapathy | Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage[END_REF]. Yu et al. observed increases in DNA strand breaks (as measured by the in vitro comet assay and the ɣH2AX immunostaining) after short-and long-term exposure (48 hours to 8 weeks) to e-cig aerosol extracts, on several normal and cancerous cell lines [START_REF] Yu | Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines[END_REF]. Finally, two studies performed on A549 and/or BEAS-2B pulmonary cells exposed at the ALI to whole smoke from reference cigarettes (M4A and/or 3R4F) reported the induction of DNA damage using the ɣH2AX assay or the in vitro comet assay [START_REF] Garcia-Canton | Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening[END_REF][START_REF] Weber | Comet assay and air-liquid interface exposure system: A new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines[END_REF].

In vivo genotoxicity studies are much less numerous and less recent. Almost all of them have been carried out on cigarette smoke and results show induction of micronuclei in bone marrow, peripheral blood and lung of exposed rodents, as well as DNA damage in lung, stomach and liver cells [START_REF] Balansky | Induction, persistence and modulation of cytogenetic alterations in cells of smokeexposed mice[END_REF][START_REF] Balansky | Less than additive interaction between cigarette smoke and chromium(VI) in inducing clastogenic damage in rodents[END_REF][START_REF] D'agostini | Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice[END_REF][START_REF] Dalrymple | Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing[END_REF][START_REF] Dalrymple | An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke[END_REF][START_REF] Nakamura | Genotoxicity-suppressing effect of aqueous extract of Connarus ruber cortex on cigarette smoke-induced micronuclei in mouse peripheral erythrocytes[END_REF][START_REF] Tice | The Influence of Antioxidants on Cigarette Smoke-Induced DNA Single-Strand Breaks in Mouse Organs: A Preliminary Study with the Alkaline Single Cell Gel Electrophoresis Assay[END_REF][START_REF] Ueno | Smoking Induces Bimodal DNA Damage in Mouse Lung[END_REF]. Some negative results have been also reported in the micronucleus and the Pig-a mutation tests. In contrast, there is very little in vivo genotoxicity data on e-cigs with only two published studies. Canistro et al. found that e-cig aerosol increased DNA damage and micronuclei formation in peripheral blood of rats exposed for 4 weeks, and the collected urine of animals induced reverse mutations in the Ames test [START_REF] Canistro | E-cigarettes induce toxicological effects that can raise the cancer risk[END_REF]. Using the 32 P-postlabeling method, Lee et al. showed that e-cig emission induced DNA adducts in lung, bladder and heart tissues of exposed mice [START_REF] Lee | E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells[END_REF].

Because of a daily and prolonged consumption of e-cig by many users, it is now essential to produce data on the mechanisms underlying the potential genotoxicity of e-cigs after long-term exposure.

In this context, the aim of our study was to investigate the in vivo genotoxic and mutagenic effects of e-cig aerosols compared to traditional cigarette smoke. After nose-only exposure of BALB/c mice, the in vivo genotoxic and mutagenic potential of smoke from conventional cigarette (3R4F) and emissions from a "Modbox" e-cig model with 0.5 Ohms coil and set at 18W (Mb18W) or 30W (Mb30W) power were assessed using (i) the in vivo comet assay in lung (primary target organ) and liver (systemic and most active metabolizer organ), (ii) the in vivo micronucleus test in bone marrow and (iii) the in vivo Pig-a gene mutation assay in peripheral blood (to identify possible systemic effects). The standard comet assay was performed within the framework of subacute (4 days), subchronic (3 months) and chronic (6 months) exposures. The micronucleus test and the Pig-a gene mutation assay, as markers of effects, were only carried out for the 3-and 6-month exposures. For ethical and scientific reasons, these three tests were applied to the same animals. In order to specifically determine oxidation-dependent DNA damage, we also measured the pulmonary 8-OHdG content after subacute, subchronic and chronic exposures, and the results were confirmed by performing a modified comet assay using the human 8-oxoguanine glycosylase (hOGG1) after the 6-month treatment.

MATERIALS AND METHODS

E-cigarettes, e-liquid and conventional cigarette

Today there is a wide variety of e-cigs and e-liquids. As explained in our previous studies [START_REF] Beauval | Influence of puffing conditions on the carbonyl composition of ecigarette aerosols[END_REF][START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF], we chose the third generation "ModBox" model, used with the "Air Tank" clearomiser equipped with a 0.5 Ω kanthal coil and with a partially closed air flow. For our experiments, we chose two power settings for the Modbox model: a "low" power of 18W and a "high" power of 30W. Both devices are from NHOSS ® (Innova, Bondues, France). For the e-liquid, we chose the best-selling NHOSS ® brand containing 65% propylene glycol, 35% glycerine, 16 mg/mL nicotine and the most common flavour, "blond tobacco", representative of a standard e-liquid in accordance with the French national organisation for standardisation (AFNOR) recommendations (AFNOR, Association Française de Normalisation, 2015). Conventional 3R4F cigarettes were obtained from the University of Kentucky (Lexington, KY, USA).

Animal model

Experiments were conducted on male BALB/c mice (Janvier Labs, Le Genest-Saint-Isle, France), 9 weeks old, 5 animals/group. This mouse strain is described as sufficiently sensitive to the chemical induction of lung cancers [START_REF] Meuwissen | Mouse models for human lung cancer[END_REF]. Animal procedures were in agreement with European directive 2010/63/EU for the protection of animals used for scientific purposes and obtained the Ethical Committee on Animal Experimentation (CEEA 75) approval.

Aerosol generation and mice exposure protocols

To avoid chemical cross-contamination, two different pieces of equipment (exposure towers and pipes) were used for e-cig and 3R4F exposures. Aerosols from e-cigs and 3R4F cigarette were generated with an InExpose e-cigarette extension system on which we adapted the Modbox and a cigarette smoking robot (SCIREQ ® , Emka technologies, Montreal, Quebec, Canada), respectively. Mice were exposed to aerosols by a nose-only tower (InExpose system, SCIREQ ® , Emka technologies). In order to perform a comparative study of the in vivo genotoxicity of the e-cig aerosols and tobacco cigarette smoke, all products were tested with Health Canada Intense puff profile (55 mL puff volume, 2 s puff duration, 30 s puff period).

Based on data from the literature and our preliminary study after a 4-day subacute exposure (data not shown), three exposure protocols were applied in this study (Table 1). First, a subacute exposure for 4 days (4 treatments at 24-hour intervals for 30, 60 or 90 min/day for both e-cigs, and for 60 min/day for 3R4F) was performed as a preliminary toxicity assessment. Then, a 3-month subchronic and a 6-month chronic exposure were realized (60 min/day, 5 days/week for e-cigs and 3R4F).

For each exposure schedule, one group was sham-exposed to fresh conditioned air (negative control). Control groups with genotoxic reference compounds were also used for in vivo genotoxicity studies (see part 2.5).

Animal body weights were recorded on Monday of each weak while clinical signs were monitored daily (data not shown).

Chemical characterization of aerosols

Chemical composition of aerosols from electronic and conventional cigarettes was assessed and described in our previous study [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF]. Chemical characterization analyses focused on the quantification of nicotine and the identification and quantification of carbonyl compounds and PAHs (see part 4).

In vivo genotoxicity assessment

The genotoxic/mutagenic potential of conventional and electronic cigarettes emissions was assessed after subacute (4 consecutive days), subchronic (3 months) and chronic (6 months) exposures by using a battery of three in vivo tests, namely the comet assay, the micronucleus assay and the Pig-a gene mutation assay. These studies were carried out using an approach very similar to that of Good Laboratory Practice (GLP). Tests, endpoints, target organs and treatment schedules are summarized in Table 1.

In vivo comet assay

The in vivo comet assay was performed in isolated lung and liver cells under alkaline conditions (pH>13) according to previously described protocol [START_REF] Platel | Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models[END_REF][START_REF] Singh | A simple technique for quantitation of low levels of DNA damage in individual cells[END_REF][START_REF] Tice | The Influence of Antioxidants on Cigarette Smoke-Induced DNA Single-Strand Breaks in Mouse Organs: A Preliminary Study with the Alkaline Single Cell Gel Electrophoresis Assay[END_REF][START_REF] Witte | Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part III: The Comet Assay as an Alternative to In vitro Clastogenicity Tests for Early Drug Candidate Selection[END_REF] and in compliance with the OECD test guideline No. 489 (OECD, 2016a). At the end of each exposure period, a positive control group was treated orally with methyl methanesulfonate (MMS) [100 mg/kg body weight (b.w)/day for 2 consecutive days in sterile water]. For all groups (i.e. treated and controls), tissues were collected once at 2-6 h after the last treatment. For the 6-month exposure time, slight modifications were added (use of hOGG1) to specifically detect oxidative DNA damage, based on Collins' and Smith's procedures [START_REF] Collins | Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA[END_REF][START_REF] Smith | hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII[END_REF]. 750 randomly selected cells per group (i.e. 50 cells per slide, 3 slides per animal, 5 animals per group) were analysed for DNA fragmentation scoring using the Comet Assay IV Image Analysis System, version 4.11 (Perceptive Instruments Ltd, Suffolk, United Kingdom). DNA damage was expressed as percentage of DNA in the tail (% tail intensity) [START_REF] Burlinson | Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup[END_REF][START_REF] Lovell | Statistical issues in the use of the comet assay[END_REF].

In vivo micronucleus test

The in vivo micronucleus test was performed in the bone marrow of treated mice in compliance with the OECD test guideline No. 474 (OECD, 2016b). A positive control group was treated orally with MMS [100 mg/kg b.w/day (x2 days) in sterile water] (see part 2.5.1). The protocol has been previously described [START_REF] Platel | Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models[END_REF]. Two slides per animal were prepared. For the determination of genotoxicity, slides were blindly scored by microscopy for the number of polychromatic erythrocytes (PCE) (2000 PCE per slide, i.e. 4000 PCE per animal) having one or more Howell-Jolly bodies (micronucleated polychromatic erythrocytes, MNPCE). For the determination of cytotoxicity, the polychromatic/normochromatic erythrocyte ratio (PCE/NCE) was determined from the microscopic examination of at least 500 erythrocytes per slide (i.e. 1000 erythrocytes per animal).

In vivo Pig-a gene mutation assay

The quantification of in vivo Pig-a (phosphatidylinositol glycan, class A) gene mutation [START_REF] Bryce | In vivo mutation assay based on the endogenous Pig-a locus[END_REF][START_REF] Dobrovolsky | The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment[END_REF][START_REF] Kimoto | Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression[END_REF] was performed with the MutaFlow PLUS Kit Mouse Blood (Litron, Rochester, New York) as previously described [START_REF] Platel | Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models[END_REF]. According to the kinetics for mutant phenotype cells appearance in circulation and ease of scoring, one month before the end of each exposure period, a positive control group was treated orally with ethyl-nitrosourea (ENU) [40 mg/ kg b.w/day for 3 consecutive days in sterile water]. Blood samples were collected after 3 and 6 months of exposure. The incidence of Pig-a mutation per animal was expressed as the number of CD24-negative red blood cells (RBC) per one million RBC, and as the number of CD24-negative reticulocytes (RET) per one million RET, using a FACSCanto II flow cytometer (BD Biosciences) running FACSDiva™ v7.0 software. The percentage of RET was also established for cytotoxicity assessment.

8-hydroxy-2'-deoxyguanosine (8-OHdG) assay

8-OHdG level was measured in the genomic DNA of mouse lung tissues following 4 days, 3 months or 6 months of exposure to either electronic or conventional cigarette aerosols. Genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) following manufacturer's recommendations. Extracted DNA was pre-treated with P1 nuclease using the reagents from Wako 8-OHdG Assay Preparation (Wako, Tokyo, Japan). This step permits to digest the DNA down to the single nucleotide level. 8-OHdG level were then determined using a competitive enzyme-linked immunosorbent assay (ELISA): Oxiselect™ Oxidative DNA Damage Kit (Cell Biolabs, San Diego, CA), according to the manufacturer's recommendations. Results were expressed as fold-change (± SD) relative to the 8-OHdG level in control mice arbitrarily set at a value of 1.

Statistical Analysis

All statistical analyses were performed with GraphPad InStat ® Software (version 3.10). For each test, differences between groups (i.e. between each concentration vs. the respective negative control) with p<0.05 were considered statistically significant.

The Mann-Whitney U-test was used for the comet assay, the micronucleus test (for the frequency of MNPCE) and the 8-OHdG content. The Student's t test was used for the statistical comparison for the PCE/NCE ratio (micronucleus test). The Dunnett's t-test (pair-wise comparison) was performed for the Pig-a gene mutation assay.

RESULTS

Results of the in vivo tests are summarized in Table 2. For each test, concurrent negative controls (animals sham-exposed to fresh conditioned air) were within the range of current observed values and concurrent positive controls induced responses that are comparable to the historical positive control data (data not shown) and produced a statistically significant increase compared with the negative control. The validity criteria for the tests were considered as fulfilled.

Subacute exposure (4-day treatment)

The genotoxic potential of electronic and conventional cigarettes was investigated in the in vivo comet assay on isolated lung and liver cells of mice after a subacute exposure (4 treatments at 24-hour intervals for 90 min/day for both e-cigs, and for 60 min for 3R4F). Results of the means of medians of percentage of tail intensity (TI) are given in Figure 1. Under tested conditions, no increase in DNA strand breaks was observed in the two selected organs, for both conventional and electronic cigarettes.

Regarding the levels of 8-OHdG in mouse lung tissues, exposure to cigarette smoke for 60 min, 4 days in a row, induced a significant increase relative to air-exposed mice (1.6 fold-change) (Figure 4A).

For e-cigs, subacute exposure to Mb18W aerosol induced no change in 8-OHdG levels regardless of the duration of exposure (i.e. 30, 60 or 90 min), whereas exposures to Mb30W emissions for 60 min and 90 min induced a statistically significant increase compared to the control (1.5 and 1.6 fold-changes) (Figure 4A).

Subchronic exposure (3-month treatment)

Results of genotoxicity/mutagenicity assessment after the 3 months subchronic exposure of mice (60 min/day, 5 days/week) are presented in Figure 2.

For both e-cigs and the conventional cigarette, no statistically significant increase in the level of DNA damage was observed, in either the liver (Figure 2A) or the lung (Figure 2B). The highest TI was obtained with Mb30W in the liver (2.3 % vs. 1.69 % for the negative control).

Regarding the frequency of MNPCE, no significant increase was found in animals exposed to Mb18W (0.75 ‰), Mb30W (1.13 ‰) or 3R4F (0.60 ‰) emissions when compared to the control group (0.55 ‰) (Figure 2C). The ratio PCE/NCE was not significantly affected by exposure to e-cig aerosols and 3R4F cigarette smoke, indicating the absence of cytotoxic effects (a very slight decrease but nonstatistically significant was observed with 3R4F).

The frequencies of mutants RET (highest value: 2.92 x 10 -6 for 3R4F) and mutants RBC (highest value: 3.42 x 10 -6 for 3R4F) did not show statistically significant increase in the animals exposed to Mb18W, Mb30W or 3R4F emissions when compared to the control group exposed to air (RET = 1.44 x 10 - 6 and RBC = 2.08 x 10 -6 ) (Figure 2D). The percentage of RET is the ratio of newly formed RNA-positive erythrocyte relative to all erythrocytes, and is used as a measure of bone marrow cytotoxicity. E-cig and conventional cigarette exposed mice did not exhibit significant changes in % RET after a 3-month exposure, thus confirming the absence of toxicity.

After 3 months of exposure to Mb30W aerosol and 3R4F smoke, 8-OHdG quantity assessment in DNA of mouse lung tissues showed a statistically significant increase compared to control (1.8 and 2.0 fold-changes, respectively) while an exposure to Mb18W emissions induced no change (Figure 4B).

Chronic exposure (6-month treatment)

Results obtained after the 6-month chronic exposure (60 min/day, 5 days/week) are presented in Figure 3.

In the standard comet assay, no increase in DNA strand breaks was observed for both conventional and electronic cigarettes. On the contrary, with the hOGG1-modified comet assay, statistically significant increases (p<0.05) in TI were observed for Mb30W and 3R4F in the liver (15.15 % and 11.46 %, respectively, vs. 1.52 % for the negative control) (Figure 3A) and in the lung (34.96 % and 30.59 %, respectively, vs. 11.57 % for the negative control) (Figure 3B), indicating oxidative DNA damage induction.

Under tested conditions, no induction of MN formation was observed in mice exposed to e-cigs 18W, 30W or 3R4F cigarette aerosols (< 0.8 ‰). No decrease of the ratio PCE/NCE was observed (Figure 3C).

No statistically significant increase in mutant frequencies of RBC (highest value: 0.37 x 10 -6 cells for 3R4F) and RET (highest value: 0.47 x 10 -6 cells for 3R4F) was observed whatever the types of cigarette compared to the control group (RET mutant frequency = 1.33 x 10 -6 and RBC mutant frequency = 0.65 x 10 -6 ) (Figure 3D). The % RET was not significantly affected indicating the absence of toxic effects in the bone marrow at this exposure level.

Consistent with subacute and subchronic exposures, a 6-month exposure to Mb18W aerosol induced no change in the level of 8-OHdG in the lung tissue DNA of mice compared to air-exposed mice, whereas Mb30W aerosol and 3R4F smoke induced a statistically significant increase (1.2-and 1.4-fold, respectively) (Figure 4C).

DISCUSSION

Electronic nicotine delivery systems are considered by public opinion to be less harmful than traditional cigarettes, and are currently used as a smoking cessation aid. Paradoxically, there is a lack of long-term in vivo studies on their health effect, thus their safety cannot be claimed. To fill this gap, we carried out a comprehensive assessment of the in vivo genotoxicity and mutagenicity of an e-cig model set to two different power levels (18W and 30W) and of conventional cigarette. The conditions of animal exposure, in terms of route (pulmonary), mode (nose-only), time (short and long-term treatment) and puff profile (Health Canada Intense profile), were designed to be as close as possible to human vaping conditions.

Under our experimental conditions, whatever the duration of animal exposure, 3R4F cigarette and e-cigs at both powers did not induce an increase in DNA strand breaks in lung and liver cells, as measured by the standard comet assay. This result may seem in contradiction with the study carried out by Canistro et al. in which e-cig aerosol produced DNA damage in leukocytes of whole-body exposed rats [START_REF] Canistro | E-cigarettes induce toxicological effects that can raise the cancer risk[END_REF]. However, as the authors themselves stated, their data should be analysed with caution as the exposure conditions used [animals were submitted to 11 cycles (puff: 6s on, 5s off, 6s on)/day, 5 days/week, for 4 weeks] did not reflect actual human exposure to e-cig aerosols. Their aim was to characterize a hazard and perhaps the use of too high doses may explain the induction of non-specific DNA damage. Other published data have also shown positive results in the in vivo comet assay on stomach, liver and/or lung with cigarette smoke [START_REF] Tice | The Influence of Antioxidants on Cigarette Smoke-Induced DNA Single-Strand Breaks in Mouse Organs: A Preliminary Study with the Alkaline Single Cell Gel Electrophoresis Assay[END_REF][START_REF] Ueno | Smoking Induces Bimodal DNA Damage in Mouse Lung[END_REF].

On the other hand, our results showed that only Mb30W and 3R4F aerosols induced a statistically significant increase in 8-OHdG formation in the lung of exposed mice after 4 days, 3 months and 6 months of exposure. At the end of our study (i.e. for the 6-month exposure) we decided to confirm this result by using a modified protocol for the comet assay. Indeed, we used the repair endonuclease hOGG1 to better characterize the mechanism of genotoxicity of e-cig emissions and conventional cigarette smoke. The hOGG1-modified comet assay is a useful tool to increase both the sensitivity and the specificity of the test and thus provide first elements of the oxidizing mode of action of test compounds [START_REF] Platel | Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels[END_REF]. The corresponding results were consistent with the 8-OHdG measurement since only Mb30W and 3R4F aerosols induced significant oxidative DNA damage in the lung and the liver of exposed mice. Our findings are also in line with our previous study [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF] and with reviews reporting that exposure to e-cig aerosols is related to oxidative stress [START_REF] Cao | Toxicity of electronic cigarettes: A general review of the origins, health hazards, and toxicity mechanisms[END_REF][START_REF] Merecz-Sadowska | A Summary of In vitro and In vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment[END_REF][START_REF] Polosa | The effect of e-cigarette aerosol emissions on respiratory health: a narrative review[END_REF][START_REF] Wang | Toxicity assessment of electronic cigarettes[END_REF]. Interestingly, Dalrymple et al. also showed, after 5 days of nose-only exposure of rats to 3R4F cigarette smoke, an increase in oxidative DNA damage in alveolar type II lung cells exclusively by using the FPG-modified comet assay (i.e. no DNA damage was observed with the classical protocol without FPG) [START_REF] Dalrymple | An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke[END_REF]. The authors also found oxidative DNA damage after 3 and 6 weeks of exposure [START_REF] Dalrymple | Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing[END_REF].

Very recently, we have carried out a comparison of the chemical composition of aerosols from Mb18W, Mb30W and 3R4F [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF]. We showed that increasing the power of the e-cig can induce an increase in the amount of toxic compounds in the aerosol (by puff, Mb18W emitted 6.9% and 51.4% less total PAHs and carbonyl compounds, respectively, than Mb30W). It has been previously demonstrated that higher power leads to higher carbonyls compounds production due to higher coil temperature (up to 300°C) and thus the thermal degradation of e-liquid and that, secondarily, the increased level of carbonyl compounds results in the formation of ROS [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF][START_REF] Geiss | Correlation of volatile carbonyl yields emitted by ecigarettes with the temperature of the heating coil and the perceived sensorial quality of the generated vapours[END_REF][START_REF] Haddad | Reactive Oxygen Species Emissions from Supra-and Sub-Ohm Electronic Cigarettes[END_REF][START_REF] Kosmider | Carbonyl compounds in electronic cigarette vapors: effects of nicotine solvent and battery output voltage[END_REF][START_REF] Zhao | Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches[END_REF]. Our results are thus consistent with these explanations since in our study oxidative DNA damage was observed only with Mb30W.

Noteworthy, we observed an almost similar response between the 3R4F cigarette and the Mb30W e-cig. It is difficult, if not impossible, to define precisely which toxic substance(s) is (are) responsible for the genotoxic effect observed in each case. The use of predictive toxicity methods (i.e. in silico models)

would be an interesting tool for this purpose as an alternative approach to experimental testing. In the study performed by Barhdadi et al., a genotoxic alert was identified by (Q)SAR models for 60 flavoring substances identified among the 129 e-liquids tested [START_REF] Barhdadi | Identification of flavouring substances of genotoxic concern present in e-cigarette refills[END_REF]. Based on information collected from EU databases 5 flavoring substances of genotoxic concern were identified (estragole, safrole, 2,5-dimethyl-4-hydroxyl-3(2H)-furanone, furylmethylketon and trans-hexenal) and 4 substances

(2,3-butanedione, 2,3-pentanedione, isoledene and β-phellandrene) gave positive result in at least one in vitro test (Ames and/or in vitro micronucleus test). Similarly, Kang et al. used (Q)SAR models to predict DNA adducts formation by flavor chemicals found in e-liquid and e-cig aerosols [START_REF] Kang | Investigating DNA adduct formation by flavor chemicals and tobacco byproducts in electronic nicotine delivery system (ENDS) using in silico approaches[END_REF].

Two chemical classes were identified, alkenylbenzenes (including estragole and eugenol) and aldehydes (including acrolein, glyoxal and methylglyoxal), well known to be produced in cigarette smoke and e-cig aerosol [START_REF] Beauval | Influence of puffing conditions on the carbonyl composition of ecigarette aerosols[END_REF][START_REF] Bekki | Carbonyl compounds generated from electronic cigarettes[END_REF][START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF][START_REF] Hutzler | Chemical hazards present in liquids and vapors of electronic cigarettes[END_REF][START_REF] Khlystov | Flavoring Compounds Dominate Toxic Aldehyde Production during E-Cigarette Vaping[END_REF][START_REF] Peace | Evaluation of Nicotine and the Components of e-Liquids Generated from e-Cigarette Aerosols[END_REF].

Lee et al., as a step towards understanding the carcinogenicity of e-cig aerosols, demonstrated that nicotine (noncarcinogenic in animals) can be nitrosated, metabolized, and further transformed into methyldiazohydroxide (MDOH) and aldehydes in lung, bladder, and heart tissues of mice [START_REF] Lee | E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells[END_REF]. They found that aldehydes and MDOH induced DNA adducts and also decreased DNA repair.

Interestingly, we previously showed that the level of nicotine delivered in the aerosols is much lower for Mb18W (60 µg/puff) than for Mb30W (137 µg/puff) and 3R4F (95 µg/puff) [START_REF] Dusautoir | Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells[END_REF].

Therefore, it can be assumed that the level of DNA adducts to be formed could be less for Mb18W which is consistent with our results.

In addition, our results revealed that both traditional cigarette smoke and e-cig aerosol induced no biologically or statistically significant increases in chromosomal aberrations and gene mutations, whatever the duration of exposure. Therefore, they are considered having no mutagenic activity under our experimental conditions. These results, although at first sight surprising, are fully in line with those of [START_REF] Dalrymple | Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing[END_REF]. In their study, rats were nose-only exposed to 3R4F cigarette (1h or 2h/day, 5 days/week) for 3 and 6 weeks. Blood was collected only at the 6-week timepoint and results showed that Pig-a gene mutations and micronucleus frequencies were not significantly increased (as mentioned above, positive results were obtained in the modified comet assay). Others have also obtained negative results in the in vivo micronucleus test on bone marrow or peripheral blood following nose-only cigarette exposure [START_REF] Schramke | Toxicological assessment of kretek cigarettes. Part 7: the impact of ingredients added to kretek cigarettes on inhalation toxicity[END_REF][START_REF] Van Miert | Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity[END_REF]. For e-cig, no data was found in the literature regarding the assessment of its in vivo mutagenicity, with the exception of the study by Canistro et al.

(2017) that showed micronuclei formation in reticulocytes of rats whole-body exposed to e-cig aerosol.

However, as explained above, their data should be compared with ours with caution because the exposure conditions they used were not intended to reflect actual human exposure to e-cig emission but rather to characterize a hazard. Indeed, in our study, the absence of mutagenic effect of reference cigarette smoke and e-cig aerosols, while it is well known that they are composed of carcinogenic substances, suggest that the experimental conditions we implemented, although realistic, may not be high enough to reach a level of exposure in bone marrow and blood to induce a positive response in the micronucleus and Pig-a tests, respectively. Furthermore, as already mentioned by [START_REF] Dalrymple | Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing[END_REF] it is possible that cigarette and e-cig do not induce mutagenic effect in organs other than the respiratory system (i.e. the first tissue of contact and target organ of tobacco products). Another important point that may explain the negative results is the sensitivity of the tests. Although the in vivo micronucleus test in bone marrow or peripheral blood is traditionally the most used in vivo test in the first instance, it is known to have a poor sensitivity of about 40-50% [START_REF] Benigni | Structural analysis and predictive value of the rodent in vivo micronucleus assay results[END_REF][START_REF] Kirkland | Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo[END_REF][START_REF] Morita | Evaluation of the sensitivity and specificity of in vivo erythrocyte micronucleus and transgenic rodent gene mutation tests to detect rodent carcinogens[END_REF]. As there is no single 'ideal' test for detecting clastogenic, aneugenic and mutagenic genetic events, it is common to use a combination of several tests (different genotoxic endpoints), as we did in our study, to increase sensitivity without reducing specificity. The Pig-a test is known for its remarkable sensitivity to mutagenic agents [START_REF] Gollapudi | The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup[END_REF] and its relatively sensitivity to clastogens [START_REF] Bhalli | Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens[END_REF].

Ideally, these tests should have been carried out on the lung and liver (i.e. on the target organs), but for methodological reasons this is not feasible. Despite an inter-laboratory study showed that the combination of the comet, micronucleus and Pig-a assays, using the same animals, may be a robust strategy for evaluating in vivo genotoxicity [START_REF] Chung | Multilaboratory evaluation of 1,3-propane sultone, N-propyl-N-nitrosourea, and mitomycin C in the Pig-a mutation assay in vivo[END_REF], it would have been relevant to perform an in vivo gene mutation test on the target organs (i.e. liver and lung) using transgenic animals. Indeed, transgenic rodent gene mutation tests have the ability to detect and quantify mutations in virtually all somatic tissues [START_REF] Gingerich | Transgenic Rodent Gene Mutation Assay in Somatic Tissues[END_REF][START_REF] Lambert | Detailed review of transgenic rodent mutation assays[END_REF]OECD, 2020). However, these tests are complex, currently expensive and not widely available.

CONCLUSION

The e-cig was initially developed as an alternative to conventional cigarette although there is insufficient data to assess its long-term safety for human health. In this context, our study was implemented with the aim of comparing the in vivo genotoxic and mutagenic potential of two low-and high-power e-cigs and the traditional cigarette, after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. In order to be as close as possible to human exposure conditions, animals were exposed to realistic doses of e-cig and cigarette emissions (i.e. Health Canada Intense puff profile) via the pulmonary route (nose-only). Under these experimental conditions, the main result of our study is that both 3R4F and Mb30W induce oxidative DNA damage in lung and liver, demonstrating that high-power ecig should be considered as "hazardous material" as traditional cigarette, whereas e-cig at low power setting seems to be devoid of in vivo genotoxic effect. These differences in results between Mb18W and Mb30W are probably attributable to lower concentrations of toxic substances (mainly carbonyls compounds) in low power e-cig aerosols, as previously described. Moreover, micronuclei and Pig-a gene mutation were not detected in reticulocytes. This suggests that our experimental conditions, although realistic, may not be sufficient to reach a level of exposure in bone marrow and blood to induce a positive response. This also raises the question of the sensitivity of these two tests in organs other than the target organ (here the lung). It is important to underline the originality of our work which is based on a complete study of the in vivo genotoxic/mutagenic potential of e-cig. Finally, our work could be completed by assessing gene mutations in the target organs (i.e. liver and lung) using the transgenic rodent mutation assay. It would also be interesting to study other non-genotoxic endpoints involved in the potential carcinogenesis of e-cig such as epigenetic alterations. All these data could lead to a better regulation of these new alternatives to conventional cigarettes. Animals were exposed to conventional cigarette (3R4F) or to e-cig (Mb18W and Mb30W) emissions for 30, 60 or 90 min/day for 4 days for subacute exposures (n = 5) and for 60 min/day, 5 days/week for 3 or 6 months for subchronic and chronic exposures (n = 8), respectively. The control group was exposed to air.

TITLE AND LEGEND OF TABLES AND FIGURES

The level of 8-OHdG is expressed as fold-change relative to the level found in control mice (± SD) measured using a competitive ELISA assay following 4 days (A), 3 months (B) or 6 months (C) of exposure. *p<0.05 (Mann-Whitney U-test).

Table 1. Summary of in vivo genotoxic/mutagenic tests performed.

For subacute exposure, mice received 4 treatments at 24-hour intervals for 90 min (and for 30 and 60 min for the 8-OHdG assay) for e-cigs, and for 60 min for conventional cigarette. For subchronic (3 months) and chronic (6 months) exposures, animals were exposed to e-cig or 3R4F cigarette emissions for 60 min, 5 times a week. X: test performed. For subacute exposure, mice were exposed 4 times at 24-hour intervals for 90 min (and for 30 and 60 min for the 8-OHdG assay) to Mb18W and Mb30W aerosols, and for 60 min to 3R4F smoke. For subchronic (3 months) and chronic (6 months) exposures, animals were exposed 60 min/day, 5 days/week, to Mb18W,

Figure 1 .

 1 Figure 1. Results of the in vivo comet assay after subacute exposure of mice to e-cig and conventional cigarette aerosols. Animals (n=5) were exposed to conventional cigarette (3R4F) smoke for 60 min/day or to e-cig (Mb18W and Mb30W) emissions for 90 min/day, for 4 consecutive days. The negative control group was exposed to air. MMS [(100 mg/kg b.w./day)x2] was used as positive control. The level of DNA fragmentation on liver (A) and lung (B) cells is expressed as the mean of medians of % of tail DNA intensity (±SD). ** p<0.01 (Mann-Whitney U-test).

Figure 2 .

 2 Figure 2. Results of in vivo genotoxicity/mutagenicity assessment after subchronic exposure of mice to e-cig and conventional cigarette aerosols. Animals (n=5) were exposed to conventional cigarette (3R4F) smoke or to e-cig (Mb18W and Mb30W) emissions for 60 min/day, 5 days/week, for 3 months. The negative control group was exposed to air. MMS [(100 mg/kg b.w./day)x2] and ENU [(40 mg/kg b.w./day)x3] were used as positive controls. (A-B) The level of DNA fragmentation is expressed as the mean of medians of % of tail DNA intensity (±SD). (C) The chromosomal aberrations frequency is expressed as the number of micronucleated polychromatic erythrocytes (MNPCE) per 1000 cells (±SD). The polychromatic erythrocytes (PCE) / normochromatic erythrocytes (NCE) ratio is used as a measure of bone marrow cytotoxicity. (D) The gene mutations frequency is expressed as the number of red blood cells (RBC) or reticulocytes (RET) per 10 6 cells (±SD). Toxicity in bone marrow was measured by % RET. * p<0.05; ** p<0.01 (Mann-Whitney U-test for the comet assay and the micronucleus test, Dunnett's t-test for the Pig-a test).

Figure 3 .

 3 Figure 3. Results of in vivo genotoxicity/mutagenicity assessment after chronic exposure of mice to e-cig and conventional cigarette aerosols. Animals (n=5) were exposed to conventional cigarette (3R4F) smoke or to e-cig (Mb18W and Mb30W) emissions for 60 min/day, 5 days/week, for 6 months. The negative control group was exposed to air. MMS [(100 mg/kg b.w./day)x2] and ENU [(40 mg/kg b.w./day)x3] were used as positive controls. (A-B)

Figure 4 .

 4 Figure 4. Results of in vivo lung 8-OHdG assessment in mice after acute, subchronic and chronic exposure to e-cig and conventional cigarette aerosols.

  FIGURE 1

Table 2 . Summary of in vivo genotoxicity/mutagenicity tests results.

 2

TABLE 1

 1

					Exposure time
	In vivo tests	End-points	Target organs
				4 days	3 months	6 months
	Standard comet assay	Primary DNA damage	Liver, lung	X	X	X
	Micronucleus test	Chromosomal aberrations	Bone marow		X	X
	Pig-a gene mutation assay	Gene mutations	Erythrocytes		X	X
	hOGG1-modified comet assay	Oxidative DNA damage	Liver, lung			X
	8-OHdG assay	8-OHdG	Lung	X	X	X

Mb30W and 3R4F aerosols. n.a.: not assessed; -: negative result; +: positive result.

FUNDING

This work benefited grant from the French Institute of Cancer (INCa): Contracts n°INCa_11505.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no conflict of interest with tobacco or e-cig industries.

ACKNOWLEDGMENTS

We thank Dr Hélène Bauderlique from the BioImaging Center Lille for facility access to the cytometer BD FACSCanto II (BICeL-campus Pasteur, 1 rue du Prof Calmette, 59019 Lille, France).

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

Anne Platel: Supervision, Conceptualization, Investigation, Funding acquisition, Visualization, Writingoriginal draft. Romain Dusautoir: Investigation, Conceptualization, Visualization, Writing -review & editing. Gwenola Kervoaze : Methodology, Investigation. Gonzague Dourdin : Methodology, Investigation. Eulalie Gateau : Methodology, Investigation. Smail Talahari : Methodology, Investigation. Ludovic Huot : Methodology, Investigation. Sophie Simar : Methodology, Writing -review & editing. Anaïs Ollivier : Methodology, Investigation. William Laine : Methodology, Investigation. Jérôme Kluza: Supervision, Conceptualization, Funding acquisition, Writing -review & editing. Philippe Gosset: Supervision, Conceptualization, Funding acquisition, Writing -review & editing. Guillaume Garçon: Supervision, Conceptualization, Investigation, Funding acquisition, Writing -review & editing. Sébastien Anthérieu: Supervision, Conceptualization, Investigation, Funding acquisition, Project administration, Writing -review & editing. Jean-Marc Lo-Guidice: Supervision, Conceptualization, Funding acquisition, Project administration, Writing -review & editing.

Fabrice Nesslany: Supervision, Conceptualization, Funding acquisition, Writing -review & editing.

Graphical abstract