Dr Yang Zhou

Dr Stéphane Maisonneuve

François Maurel
email: maurel@u-paris.fr

Juan Xie
email: joanne.xie@ens-paris-saclay.fr

Dr Rémi Métivier
email: remi.metivier@ens-paris-saclay.fr

Prof J Xie

Prof F Maurel

Competitive Photoisomerization and Energy Transfer Processes in Fluorescent Multichromophoric Systems

Keywords: Fluorescence, Photoisomerization, Energy transfer, Multichromophoric systems, Molecular Dynamics

Multichromophoric systems showing both fluorescence and photoisomerization are fascinating, with complex interchromophoric interactions. The experimental and theoretical study of a series of compounds, bearing a variable number of 4dicyanomethylene-2-tert-butyl-6-(p-(N-(2-azidoethyl)-N-methyl) aminostyryl)-4H-pyran (DCM) units are reported. The photophysical properties of multi-DCM derivatives, namely 2DCM and 3DCM, were compared to the single model azido-functionalized DCM, in the E and Z isomers. The (EE)-2DCM and (EEE)-3DCM were synthesized via the click reaction. Steady-state spectroscopy and photokinetics experiments under UV or visible irradiation indicated the presence of intramolecular energy transfer processes among the DCM units.

Homo-and hetero-energy transfer processes between adjacent chromophores were confirmed by fluorescence anisotropy and decays. Molecular dynamics simulations for 2DCM were carried out and analyzed using a Markov state model, providing geometrical parameters (orientation and distance between chromophores) and energy transfer efficiency. This work contributes to a better understanding and rationalization of multiple energy transfer processes occuring within multichromophoric systems.

Introduction

Multichromophic molecules and assemblies are envisioned as a promising category of functional building blocks to be applied in light-harvesting materials, optoelectronic devices, adjustable polymers, or supramolecular systems. [1] Multichromophores containing a large number of chromophores enable specific features in a wide range of fields. For example, the multichromophores including photochromic moieties (multiphotochromic compounds) and fluorophores can exhibit photoswitchable colors, high-contrast fluorescence via resonance energy transfer (RET), and they can serve as multidimensional logic gates or induce multiple states supramolecular structural rearrangements. [1c, 2] Besides applications involving structural changes or multi-logic gates, several reports highlight the use of photoisomerizable units, such as azobenzenes, in multichromophoric systems to enable non-linear optical features. [3] Some studies were reported about photoisomerizable chromophore-chromophore interactions, whereas most of them focus on the changes in the mechanical forces of films or crystalline solids, or the electronically or conjugationally influencing structures. [4] As far as we know, photoisomerizable fluorophores included in large multichromophoric systems have not been explored to date. 4-Dicyanomethylene-2-tert-butyl-6-(p-(N-(2-azidoethyl)-Nmethyl)aminostyryl)-4H-pyran (DCM) derivatives represent one category of photoisomerizable fluorophores with a donor-acceptor character, as previously reported by our group. [5] The double-bond isomerization can be induced by light, leading to the interconversion between the emissive E-form and the nonemissive Z-form. Several DCM analogues have been investigated, [5] demonstrating their efficiency, fatigue resistance, and ability to serve as brightly emissive molecular photoswitches. Since the DCM molecule shows both fluorescence and photoisomerization properties, we could expect interesting interactions by Förster resonance energy transfer (FRET) processes and following, novel optical features, when merged together in larger molecules. Therefore, we designed a series of DCM-based molecules, from the single model chromophore, DCM, to more sophisticated structures, 2DCM and 3DCM, where two and three parent azido DCMs are connected together via a central flexible dendritic linker by the cooper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. [6] The corresponding chemical structures are shown in Figure 1, where all the DCM units are represented in the E-form. Such a design strategy offers the possibility to consider further functionalization with other molecular systems. In this work, we report on the synthesis, photophysical and photochemical investigations of the 2DCM and 3DCM multichromophoric compounds, which are compared to the DCM model chromophore, by means of steady-state absorption spectroscopy, time-resolved fluorescence, fluorescence anisotropy, and photokinetics experiments. The photophysical properties of the series were investigated, and the comparison of their respective photokinetics under UV and visible irradiation could reveal the interchromophoric interactions existing among the DCM units. At the theoretical level, molecular dynamics (MD) simulations were performed on the 2DCM molecule, which is a well-representative derivative to investigate both homo-FRET and hetero-FRET processes, followed by Markov state model (MSM) implementation to unravel the correlation between the molecular conformational changes and their impact on the FRET efficiencies.

Results and Discussion

Synthetic protocols and molecular structures

The design of functional dendritic architectures with well-defined structures is a very active an exciting field of research, and many multichromophoric systems have been synthesized with dendrimers or dendrons bearing large numbers of chromophores. [7] With the aim of gathering several independent DCM units, two dendritic linkers 2 and 3 with two and three propargyl groups respectively were firstly prepared (Scheme 1). Compound 2 was obtained after di-O-propargylation of isopropylidene acetal protected pentaerythritol 1, [8] while 3 was synthesized according to the reported procedure. [9] CuAAC reaction of 2 with (E)-DCM under microwave irradiation led to 2DCM as pure (EE)-isomer in 82% yield, by shielding the ambient light during the whole synthesis procedures. For the synthesis of 3DCM, compound 3 was firstly reacted with (E)-DCM under microwave activation, followed by azidation with sodium azide in the presence of catalytic amount of sodium iodide in DMF at 80 °C. the (EEE)-3DCM was obtained in 37% total yield from 3. Scheme 1. Synthesis of (EE)-2DCM and (EEE)-3DCM.

(E)-DCM was an orange red crystal, whereas (EE)-2DCM and (EEE)-3DCM were obtained as red solids. The X-ray diffraction structure of DCM single crystals revealed its s-trans-(E) configuration, as already described in our previous studies. [5b] The s-trans-(E)-DCM is the most stable form among its four identified isomers and conformers, with a planar structure. However, the crystalline ability of (EE)-2DCM and (EEE)-3DCM is not good enough to allow single crystal X-ray diffraction analyses. When 2DCM and 3DCM are synthesized, we assume that the DCM units adopt preferentially the s-trans-(E) configuration, with high degrees of freedom between the DCM units allowed by the flexible central dendritic linker.

Steady-state spectroscopy and photokinetic studies

According to our previous reports, THF was selected as the ideal solvent, since it allows the highest photoisomerization conversion of the DCM chromophore under irradiation, with significant emission properties. [5a] The absorption and fluorescence spectra of DCM, 2DCM and 3DCM in their E forms in THF are depicted in Figure 2. The absorption and fluorescence show that the three compounds have similar absorption and emission bands, with their maxima located at 457 nm and 588-590 nm, respectively. Table 1 presents their molar absorption coefficient () at 457 nm, with increasing values in the sequencial order DCM < 2DCM < 3DCM. However the  increments are not exactly proportional to the number of DCM units: it is only 1.8 times higher (2.6 times, resp.) higher for 2DCM (3DCM, resp.) than for DCM. The deviation from linearity of  with the number of DCM units indicates that interchromophoric interactions between neighbouring chromophores exist in the multichromophoric systems. In addition, the fluorescence quantum yield (F) was measured at 0.25 for DCM, but slightly decreases to 0.21 for 2DCM and 3DCM. Again, the decrease of F in diluted THF may be due to some level of intramolecular - stacking between the DCM units within the 2DCM and 3DCM. It is worth noting that the DCM chromophores are only fluorescent in their E form, not in their Z form. [5] After irradiation at 485 nm (inducing the EZ forward reaction of DCM) and 335 nm (to induce the ZE backward reaction), photostationary states (PSS) were obtained for the three compounds, showing a decrease of the main absorption band at 457 nm and a clear isosbestic point at 391 nm (Figure 2), revealing that the DCM units can be considered as simple photoswitches, either in their E or Z forms. However, major differences on the PSS photoconversion levels reached at 485 nm and 335 nm were identified through their absorption spectra. The absorbance ratio of the two PSS at 485 nm and 355 nm, APSS-485/APSS-335, measured at 457 nm, is relatively low for DCM (0.75) which reflects its noticeable EZ conversion under visible irradiation. Interestingly, the 2DCM is less prone to be isomerized to the Z-form under 485 nm irradiation (APSS-485/APSS-335 = 0.90), whereas 3DCM shows the lowest photoconversion (APSS-485/APSS-335 = 0.96). Obviously, the number of DCM units has a great impact on the E-Z interconversion by light. [a] Emission quantum yields F were determined using Coumarine 153 in ethanol (Φem = 0.54) [10] as the standard, exc = 435 nm.

To further investigate the photoisomerization properties, we carried out photokinetic experiments under similar irradiation conditions for DCM, 2DCM and 3DCM, starting from the fresh Eforms. Each compound was dissolved in THF solution, continuously probed by absorption spectra, and irradiated with visible light at 485 nm under vigorous stirring, which induced the EZ photoisomerization of the DCM units to reach the PSS485. After 45 min, a subsequent irradiation at 335 nm was applied to trigger the reverse ZE tranformation to the PSS335. The absorbance was monitored at 457 nm and corresponding timeprofiles are plotted in Figure 3. During the first sequence of irradiation at 485 nm, 3DCM reaches the PSS equilibrium first (c.a. 10 min) and shows a limited decrease of absorbance (Abs < 0.06), whereas DCM presents an opposite behaviour, with as a large absorption change (Abs > 0.2) but a slow photokinetics, the PSS equilibrium not being reached after 30 min of irradiation. 2DCM exhibits intermediate properties, with a PSS reached in 20 min with an absorbance decrease of Abs  0.1. During the second sequence of irradiation at 335 nm, the situation is similar, with a slow (resp. fast) photokinetics associated to large (resp. small) absorption changes for DCM (resp. 2DCM and 3DCM). This intriguing observation that the photoconversion rate and yield evolve in opposite ways, with a strong dependence on the number of DCM units in the molecules, could originate in the possible energy transfer processes that can act within chromophores in the 2DCM and 3DCM cases. As a matter of fact, spectral overlaps exist between emission and absorption spectra of DCM, suggesting the possibility of intramolecular FRET between neighbouring chromophores in the case of 2DCM and 3DCM derivatives. More specifically, Figure 4 overlays the emission spectrum of (E)-DCM with the absorption spectra of (E)-DCM and (Z)-DCM, revealing a small but significant spectral overlap in both cases (highlighted in red in Figure 4). Therefore, two distinct energy transfer paths can be expected in 2DCM and 3DCM: homo-FRET processes between two (E)-DCM chromophores, and hetero-FRET processes between DCM units from the E form (donor) to the Z form (acceptor). As illustrated in Figure 4, when mutual orientations and interchromophoric distances are favorable, both processes can occur in the multichromophoric systems 2DCM and 3DCM.

In the case of 3DCM, the mixed model with two types of homo-FRET and hetero-FRET occurring simultaneously is also possible. Such intramolecular FRET pathways could explain the differences observed in converison yields and rates for the three DCM-based compounds. Indeed, hetero-FRET may represent an efficient pathway leading to the deactivation of the E form and promoting the formation of Z form excited states, which in turn would decrease the EZ reaction efficiency and favor the ZE reaction. Therefore, hetero-FRET processes are expected to decrease the EZ conversion yield under visible irradiation, but also increase the rate to reach the PSS equilibrium, through enhanced ZE rates. This effect would be consistently higher in 3DCM, where multiple FRET pathways are foreseen, compared to 2DCM. In the case of 3DCM, homo-FRET between identical DCM units in the E form (hopping mechanism), can increase the probability to find an energy transfer route from the E forms to the Z form (e.g. EEZ stepwise energy transfer sequence), thus indirectly promoting the excitation of the Z form.

Fluorescence decays and anisotropy investigations

Fluorescence decay curves of the DCM, 2DCM and 3DCM were recorded by the time correlated single photon counting (TCSPC) method (Figure 5), with an excitation (resp. emission) wavelength at 400 nm (resp. 590 nm). The emission decays of the three compounds are not perfectly overlapping, with a more pronounced multiexponential character for 2DCM and 3DCM.

The decay curves were fitted by a global analysis procedure using a bi-exponential function for the case of DCM, and a triexponential function for 2DCM and 3DCM, with satisfactory  2 R < 1.2. According to our previous studies, [5] the emission arises only form the (E)-DCM units. Only two components 1 = 0.25 ns and 2 = 0.84 ns were identified for DCM can be ascribed to different conformers of the E form, namely the s-trans-(E)-DCM (longer decay-time 2) and s-cis-(E)-DCM (short decay-time 1). [5] An additional third component 3 = 1.90 ns was found for 2DCM

and 3DCM, associated with small contributions to the whole decays (a3 < 0.07, f3 < 0.18), which can be ascribed to a certain proportion of DCM units interacting together, through intramolecular - stacking for instance, as already suggested in the previous paragraphs. Such interactions may restrict nonradiative deactivation pathways of the DCM units, resulting in a longer decay time. After irradiation at 485 nm, the decay curve of DCM remained unaffected, whereas 2DCM and 3DCM showed slightly shortened fluorescence decays: the preexponential coefficient and the fraction of fluorescence intensity of the shortest decay-time 1 (a1, f1) increased, while those of 2 (a2, f2) decreased, and the proportion of the third decay component 3 remained almost stable. For 2DCM and 3DCM, the changes in relative weights of 1 and 2, corresponding to the s-cis and s-trans conformers of the (E)-DCM units, respectively, can be tentatively explained by the conformational redistribution of the chromophores following the relaxation process after excitation. Since constraints from the linkers exist for the DCM units in 2DCM and 3DCM, the s-ciss-trans interconversion after the photoreaction is probably hindered by the covalent connections, which could lead to higher proportions of s-cis-(E) species. Unfortunately, the homo-FRET and hetero FRET could not be confirmed by time-resolved fluorescence because: (i) homo-FRET is basically not expected to affect the fluorescence decays of the (E)-DCM, and (ii) the (Z)-DCM being non emissive, the hetero-FRET from (E)-DCM to (Z)-DCM would induce a fluorescence quenching and a strong decrease of (E)-DCM lifetime, down to a level below the temporal resolution of our instrumental setup (0.03 ns). Steady-state fluorescence anisotropy being a relevant method to investigate the homo-FRET in multichromophoric assemblies, [11] it was measured for DCM, 2DCM and 3DCM derivatives in propylene glycol at -45 °C (Figure 6), which forms a vitrified medium and freezes the molecules to avoid rotation movements during the excitation timescale. The steady-state anisotropy (𝑟̅ )

of DCM reaches a level as high as 0.36 for exc > 410 nm, which is very close to the maximum theoretical limit of the singlephoton excitation fluorescence anisotropy r0 = 0.4. This high value reflects the almost colinear absorption and emission transition dipole moment of DCM: the angle  between these transition dipole moments was calculated to be 15° for DCM. At exc < 410 nm, the anisotropy decreases down to almost zero at 360 nm, which is consistent with transition dipole moments of higher excited states. 2DCM and 3DCM show a steady-state anisotropy plateau value of 0.22 and 0.18 in the range 430 nm < exc < 500 nm (Figure 6). This depolarization compared to DCM clearly confirms that homo-FRET processes do occur in 2DCM and 3DCM, the latter being more depolarized in the same experimental condition. At longer excitation wavelength (exc > 500 nm), the steady-state anisotropy rises up to 0.35 and 0.30 for 2DCM and 3DCM, respectively, due to the so-called Weber's red-edge effect: excitation in the low-energy region of the S0S1 electronic transition selects the DCM chromophores which are less prone to EE homo-FRET, and as a result, leads to an increase of the 𝑟̅ value. Therefore, steady-state anisotropy provides evidences of homo-FRET within DCM units in 2DCM and 3DCM, and we can assume that the higher depolarization of 3DCM emission is related to larger homo-FRET efficiencies, due to more favorable orientation arrangements of the chromophores and/or shorter interchromophoric distances. The transition dipole moment orientations of the DCM units within multichromophoric architecture, and inter-DCM distance parameters are critical parameters influencing the efficiency of homo-FRET and hetero-FRET in these systems. However, direct and dynamic measurements of such structural parameters are out of reach by conventional experimental techniques. For this reason, we decided to perform MD simulations to characterize the geometrical parameters and their influence on the FRET efficiencies.

Modelling of the FRET parameters of 2DCM

To examine the geometrical parameters of the DCM-DCM interaction and their effect on the homo-FRET and hetero-FRET efficiencies, we selected 2DCM as the simplest molecule to be studied by MD simulations. Two models can be prepared, representing the typical FRET patterns in dendritic multichromophoric DCM-based molecules. The first model is the (EZ)-2DCM configuration, where we assign the (E)-DCM as the donor and the other (Z)-DCM as the acceptor in the frame of the EZ hetero-FRET. Besides, the (EE)-2DCM represents a good model to investigate the EE homo-FRET (the donor and acceptor chromophores are chosen arbitrarily).

Due to the large size of the studied systems, the initial structures for MD simulations were optimized using the mixed ONIOM method. The DCM units were calculated using PBE0 functional and the 6-311G+(d,p) basis set while the bridge connecting the DCM unit was treated using the semi-empirical PM6 method (see details in Figure S1). The optimized geometries have been built and converged in an unfolded conformation for both (EZ)-2DCM and (EE)-2DCM structures, as shown in Figure 7 and Figure S1. They were packed with 1500 THF molecule by Packmol (Figure S2), [12] and MD simulations were performed with the GROMACS software package version 2020 [13] using general AMBER force field (GAFF) [14] from AmberTools. [START_REF] Case | Amber 2021[END_REF] All the simulations for (EZ)-2DCM and (EE)-2DCM were performed in a sequence involving the energy minimization, the equilibrium isothermal-isobaric (constant particle number, pressure and temperature, NPT) ensemble, and the production NPT ensemble for 200 ns, which was validated by comparison with longer trajectories (Figure S5 and Table S1).

MD simulations and FRET efficiency trajectories.

A first observation to the MD trajectories showed us that the DCM unit keep their E or Z isomeric state over the whole simulation, but could flip sometimes from s-cis to s-trans and vice versa (Figure S3). The instantaneous values RDA(t) of the distance between the donor-acceptor pair and orientation factor  2 (t) were generated by the MD software toolkit from the trajectories. In this MD section, even though energy transfer processes involve the donor species in the excited state, we assume that the ground state geometry of the multichromophoric systems can be used to estimate the FRET efficiency EFRET, by the equation ( 1):

𝐸 𝐹𝑅𝐸𝑇 = 1 1+(𝑅 𝐷𝐴 𝑅 0 ⁄ ) 6 (1)
where R0 is the Förster radius of the donor-acceptor pair, calculated by the equation ( 2):

𝑅 0 6 = 9000×ln (10)×𝜅 2 ×Φ 𝐷 0 128×𝜋 5 ×𝑁 𝐴 ×𝑛 4 × 𝐽(𝜆) (2)
where n is the refractive index of THF, D 0 is the fluorescence quantum yield of the DCM chromophore in absence of energy transfer, J() refers to the absorption and emission spectral overlap integral, NA is the Avogadro constant. The initial geometries of (EZ)-2DCM and (EE)-2DCM are depicted in Figure 7 and Figure S1. The RDA was derived from the distance between atoms closest to the mass centers of each DCM fragment, and the transition dipole moment vectors of the (E)-DCM and (Z)-DCM fragments at their first excited states were calculated by TDDFT. The  2 (t) was then calculated through the equation (3): 𝜅 2 = (sin 𝜃 𝐷 sin 𝜃 𝐴 cos 𝜑 -2 cos 𝜃 𝐷 cos 𝜃 𝐴 ) 2

(3)

where D, A, and  are defined in Figure 7. The (Z)-DCM molar absorption coefficient was determined from the DCM conversion rate 𝛼 𝑍 , by a well-established method in our group. [5] From the DCM spectra shown in Figure 4a, the spectral overlap intergrals J() were then calculated for (EZ)-2DCM and (EE)-2DCM to be 1.0310 13 nm 4 M -1 cm -1 and 5.9010 13 nm 4 M -1 cm -1 , respectively. The instantaneous EFRET(t) can be derived from the instantaneous orientational factor  2 (t) and the interchromophoric distance RDA(t) (corresponding histograms and average values in Figure S4). 3 nm, pushing the FRET efficiency up to nearly 100%, except for some extremely unfavorable orientations. For RDA < 1 nm, the conformations of (EZ)-2DCM are mostly in a folded geometry, as exemplified for the geometry #3 in Figure 8f, with  2 close to 1 (two transition dipole moment almost parallel to each other). When the interchromophoric distance is in the interval 1.5 < RDA < 2 nm, i.e.

58.5-61.0 ns in Figure 8b-c, the  2 starts to control the EFRET level, the higher the  2 , the higher the EFRET. The geometry #1 displayed in Figure 8f presents a folded structure, but an unfavored orientation of the DCM units ( 2  0) leading to an absence of hetero-FRET. When the RDA becomes higher than 1.5 nm, the  2 fully dominates the FRET efficiency shape: the EFRET trajectory synchronizes the  2 one in Figure 8b-c The unfolded conformation #2 shown in Figure 8f highlights the typical situations where both large DCM-DCM distance and almost perpendicular orientation prevent the FRET process.

In the case of (EE)-2DCM, the MD simulation provides similar trajectories (see Figure S6), with even higher levels of homo-FRET efficiency throughout the whole trajectory length, resulting in an average EFRET value as high as 87.8%. In a first approach, the larger homo-FRET efficiency compared to hetero-FRET efficiency is essentially due to the higher spectral overlap, J() being almost six times larger (vide supra). From the histogram shown in the Figure S6, most frames contribute to EFRET over 90%. The enlarged areas of the (EE)-2DCM trajectory highlight that the orientations and distances of the DCM fragments are rarely unfavorable to the homo-FRET. Several examples of conformations have been selected in Figure S6, and even the unfolded form can reach an EFRET value of 55% since the transition dipole moments are favorably parallel.

Concerning the interchromophoric distance between the DCM units, RDA, the Table 3 lists three categories, counting the situations (frames) where RDA  1, 1< RDA <2 and RDA  2. It provides a synthetic picture of conformations in the two trajectories. The RDA  1 region of (EZ)-2DCM reaches 91.5% average EFRET and reaches 98.5% in the case of (EE)-2DCM. This region can be viewed as mostly dominated by the interchromophoric distance, since even unparallel orientations are still favorable to FRET processes. In the intermediate 1 < RDA < 2 region, the average EFRET of (EZ)-2DCM decreases substantially (64.7%) whereas it remains at a high value in the case of (EE)-2DCM (92.3%). When RDA  2, the average EFRET drops down in both cases (23.2% and 61.5% respectively), and the  2 starts to control the EFRET level, as described previously.

Therefore, the MD simulations confirm that hetero-FRET and homo-FRET processes can take place within chromophores of (EZ)-2DCM and (EE)-2DCM, and the trajectory-based instantaneous FRET analysis reveals that the completely folded conformation is the most favorable to FRET, which can reach almost 100%. Based on the above statements, we further continued statistical implementations on the MD simulation data to identify the most representative geometries using the principal component analysis (PCA) method (Figures S7-8). More interestingly, connections between the main conformations found by PCA of (EZ)-2DCM and (EE)-2DCM, can be revealed by the Markov State Model (MSM) method, as detailed below. Markov State Models (MSMs) of 2DCM. To further rationalize the conformational changes occuring during the trajectories produced by MD simulations, we built the MSM for both trajectories, corresponding to (EZ)-2DCM and (EE)-2DCM. The MSM reveals the important intermediate states from the MD simulation data and their mutual relationships. The analysis was carried out using the PyEMMA package. [START_REF] Scherer | [END_REF] The MD trajectories were taken into a time-lagged independent component analysis (tICA), and featurized by the software. The tICA maximizes the autocorrelation of the linearly transformed coordinates by a lag time , finding a maximally slow reaction in the MD simulation corresponding to the lag time of the input parameter as the first independent component (IC). This method allows to identify all conformational components even with insufficient sampling. A lag time of 1 ns was selected for each trajectory (for time lags longer than 1 ns, the timescale curves level off, for both (EZ)-2DCM and (EE)-2DCM, as shown in Figure S9). Furthermore, the Chapman-Kolmogorov tests for the two MSMs show that the lag time τ = 1 ns model accurately predicts the behaviors at longer timescales. The resulting 2D and 3D free energy landscapes (FELs) of (EZ)-2DCM and (EE)-2DCM configurations along their first and second independent components are shown in Figure 9. The principal component analysis (PCA) was carried out, as shown in the Figures S7-8. The 200 ns trajectories for both 2DCMs have rather low cosine contents of the first two principal components (PCs): (EZ)-PC1 = 0.03, (EZ)-PC2 = 0.06, (EE)-PC1= 0.16, (EE)-PC2 < 0.01, which indicates an acceptable sampling of this duration. The MSMs were obtained with 200 microstates using κ-means clustering method and further lumped into 6 metastable states using the Perron cluster analysis (PCCA+) method, respectively. These metastable states were clustered to extract the major conformations of (EZ)-2DCM and (EE)-2DCM, as depicted in Figure 9a,c with several colors. The locations of the representative conformations from the metastable states are marked on the 3D FELs. The MSMs with transition pathways and pathway percentages on 2D FELs are displayed in Figure 9b,d. In the (EZ)-2DCM MSM (Figure 9a-b), the main transition pathway starts from the unfolded form EZ-A, transits through the completely folded EZ-3 and EZ-4, and finally ends with the more compact rolled EZ-B, with a total pathway percentage of 75%. Another pathway, counting for 21%, omits the EZ-3, getting twisted conformations directly (EZ-2) and then converges into EZ-4 and EZ-B. We can clearly observe that this is a process which continuously shrinks the interchromophoric distance. The EZ-A unfolded types generally correspond to RDA > 2, whereas the most compact EZ-1 and EZ-B have RDA < 1, as visualized in the last section, and the intermediates have geometries with 1 < RDA < 2 frames. Consequently, the average FRET efficiency increases along the pathways. The other minor routes with lower fluxes follow the same pattern as well, with decreasing RDA from the unfolded conformations, thus improving the EFRET along the path. On the (EE)-2DCM side (Figure 9c-d), the situation is different. The unfolded forms EE-3 are the most probable intermediates during the process, and the MSM indicates a "folded conformations"  "unfolded conformations"  "single folded conformations" chain. The single folded conformations (EE-2 or EE-4) converge into the EE-B, for about 83% of the pathway flux. The pathways are based on the shift of the single folded and folded conformations via the unfolded conformations. Besides, other paths omitting the unfolded EE-3, directly through EE-2 and EE-4, can occur as well. In addition, 8% pathway flux is induced by the rolled folded types from the starting EE-A. We explored the MD simulation trajectories with both MSM (tICA) methods (and PCA, see Figures S7-8), extracting the pathways along the typical conformations of 2DCM. The representative conformations in the FELs can be categorized according to their RDA, which are in turn related to the FRET efficiencies. In the case of (EZ)-2DCM, the evolution process from the unfolded forms to the completely/rolled folded conformations results in nearly 70% enhancement of the FRET efficiency. (EE)-2DCM undergoes an increase of FRET efficiency by 30% as long as its RDA starts to shrink. Connected to the fluxes and pathway percentages of MSMs, the conformational fluctuation causing FRET efficiency variations in THF solution can be deduced.

Conclusion

We have synthesized and characterizeda series of DCM-based molecules, namely DCM, 2DCM and 3DCM, to serve as first specimens and models of photoisomerizable multichromophoric assembies, and to investigate their behaviors under irradiation. Although possessing very similar absorption and emission spectral features, lower photoconversion yields and faster photokinetics were observed for 2DCM and 3DCM compared to DCM, which originate from intramolecular energy transfer processes. The homo-FRET was demonstrated to occur, By means of steady-state fluorescence anisotropy, an increasing depolarization of the emission was observed for 2DCM and 3DCM, demonstating the occurence of homo-FRET within the systems. Although time-resolved fluorescence cannot determine precisely the hetero-FRET, we selected the 2DCM molecule as a model to carry out MD simulations in two different states, (EZ)-2DCM and (EE)-2DCM. Thanks to the MD simulations, we could rationalize the connections between the FRET efficiency, the interchromophoric distances, orientation factors, explaining the reasons of the FRET fluctuations in time. Statistically, higher efficiencies FRET were identified for homo-FRET (87.8% average) than for hetero-FRET (63.4% average). The MSMs methodology and the statistics based on FELs, related to the MD simulations data, provided the main transition pathways among the structural conformations. In summary, we elucidated the strong FRET dependence on the molecular conformational changes. We can conclude that the photoisomerizable multichromophoric molecular architectures based on DCM chromophores are prone to be strongly affected by intramolecular energy transfer processes (homo-and hetero-FRET), with tuneable properties under irradiations. The future designs on multi-DCM systems should take into account the structural and geometrical factors (distances, orientations) to achieve desired performances.

Experimental Section

Materials. The synthetic protocols of DCM, 2DCM and 3DCM are given in the Supporting Information. All commercially available compounds and solvents are purchased from Sigma-Aldrich affiliated to Merck KGaA, Carlo Erba Reagents, TCI chemicals and Acros Organics affiliated to Thermo Fisher Scientific without further purifications. 1H-, 13C, COSY, HSQC NMR data were acquired by JEOL ECS-400 spectrometer with a broadband probe. Spectroscopic measurements were performed with spectroscopic grade solvents from Carlo Erba Reagents and Sigma-Aldrich affiliated to Merck KGaA.

Steady-state spectroscopy. Absorption spectra at room temperature were recorded using Agilent Cary-4000 or Cary-5000 UV spectrometers. Fluorescence emission and anisotropy data were measured with Horiba Jobin-Yvon Fluorolog-3 spectrofluorometer equipped with polarizers in excitation and emission. Quartz cuvettes with a 1 cm path length were used. Fluorescence emission quantum yields were determined using Courmarine 153 in ethanol (Φem = 0.544) as the reference. Steady-state anisotropy was recorded by means of the IVV, IVH, IHV and IHH measurements (V, vertical; H, horizontal) of the sample at -45 °C in propylene glycol, after calibration of the polarizers with a Ludox scattering suspension.

Photoisomerization studies. Irradiation experiments were performed at room temperature, with a Hamamatsu Hg-Xe lamp LC8 (Lightningcure 200 W) excitation source. The desired wavelengths were selected using appropriate Semrock interferential filters at 335 nm and 485 nm. The incident lamp power was measured by means of an Ophir PD300-UV photodiode, subtracting the residual NIR contribution from the total value. Photoisomerization kinetics were determined using a home-made setup allowing the collection of absorption spectra at high rates under continuous irradiation. A Xenon lamp (75 W) was used as a probe light source placed at 90° with respect to the irradiation beam. Spectra were recorded every 0.2 seconds, with a spectrometer coupled with a CCD (Roper Scientific and Princeton Instruments, respectively). Time-resolved fluorescence spectroscopy. Fluorescence decay curves were obtained by the time-correlated single-photon counting (TCSPC) method. The setup is composed of a titanium sapphire Ti:Sa oscillator (Spectra Physics, Maï Taï) emitting pulses of 100 fs duration for the fundamental laser beam, at 80 MHz frequency. The repetition rate is reduced down to 4 MHz by a pulse picker which implements an acoustooptic modulator. Non-linear SHG crystals generate the desired wavelength (GWU Lasertechnik, UHG-23-PSK), then the beam is directed to the sample solution after adjusting the excitation power with an intensity attenuator filter wheel. Fluorescence photons are detected at 90° through a polarizer at the magic angle and a monochromator, by means of a micro-channels plate photomultiplier (MCP-PMT R3809U-50, Hamamatsu), connected to a TCSPC module (SPC-630, Becker & Hickl). Fluorescence decays were finally processed with the help of the global non-linear least-squares minimization method including reconvolution analysis (Globals, Laboratory for Fluorescence Dynamics at the University of California, Irvine).

Quantum chemical geometry optimizations. The DCM and DCM units in the 2DCM and 3DCM were optimized by density functional theory (DFT) by using Gaussian 16 software package. [17] The 2DCMs were optimized by the ONIOM strategy in Gaussian 16, partitioning DCM units into DFT and the dendritic linker part into PM6 semi-empirical functional. PBE0 was used for all DCM and DCM units at 6-311G+ (d,p) basis set level. IEFPCM solvent model of THF is used. The optimized geometries were taken into the MD simulation input set-ups.

Molecular Dynamics (MD) simulations. The MD simulation inputs were based on the geometries optimized in quantum chemistry level. The electrostatic potentials of 2DCMs were calculated at B3LYP/def2-SVP level of theory by following the restrained electrostatic potential (RESP) procedure. Each configuration of 2DCM was encapsulated with 1500 THF molecules by Packmol program, [12] setting the mutual distance tolerance limit at 2 Å, and modelled by AmberTools [START_REF] Case | Amber 2021[END_REF] with general AMBER force field (GAFF). [14] The MD simulation was carried out using GROMACS software package version 2020. 2 [13] trajectories of (EE) and (EZ) configured 2DCM were obtained. All trajectories were written every 5 ps for analysis. The production ensembles use the leapfrog integrator with the step size of 2 fs, a reference temperature at 298 K, a reference pressure at 1 bar.

Molecular Dynamics (MD) analyses. The 2DCM trajectories were pretreated by GROMACS toolkit (principal components analysis, PCA). The covariance matrix and following eigenvalues, eigenvectors were generated and calculated by GROMACS toolkit or PyEMMA. The alltrajectory clustering was realized in VMD. [18] The MSM was obtained from the MD simulation data by analyzing with program PyEMMA. [START_REF] Scherer | [END_REF] The MD simulation data of 2DCM coordinates was estimated by computing a time-lagged independent component analysis (tICA). The Markov State Model (MSM) was validated by the methods provided from PyEMMA, including the implied timescale test and Chapman-Kolmogorov test (Figure S10). The free energy landscapes (FELs) were drawn in Wolfram Mathematica. After getting the probability density P(x), we apply the equation below to calculate the Gibbs free energy with Boltzmann constant kB.

Table of Content

Multichromophoric systems showing both fluorescence and photoisomerization were investigated by experimental and theoretical methods. We highlight the prominent role of intramolecular homo-and hetero-energy transfer processes between the different isomers of the chromophores at the origin of their peculiar photophysical and photochemical properties.

Figure 1 .

 1 Figure 1. (a) E-Z photoisomerization reaction of DCM and (b) structures of 2DCM and 3DCM derivatives with each DCM unit being in the E form.

Figure 2 .

 2 Figure 2. Absorption spectra after irradiation at 485 nm (dashed lines, PSS485) or 335 nm (full lines, PSS335) and emissionspectra before irradiation (dotted lines) of DCM (pink color) 2DCM (blue color) and 3DCM (orange color), in THF.

Figure 3 .

 3 Figure 3. Absorbance at 459 nm of DCM (pink color), 2DCM (blue color) and 3DCM (orange color) in THF, as a function of irradiation time at 485 nm and 335 nm.

Figure 4 .

 4 Figure 4. (a) Absorption spectra of (E)-DCM and (Z)-DCM, and emission spectrum of (E)-DCM. Spectral overlaps are highlighted in dark and light red colors. (b) Illustration of E↔Z photoisomerization of DCM and intramolecular energy transfer processes (homo-FRET and hetero-FRET) occuring in 2DCM and 3DCM.

Figure 5 .

 5 Figure 5. Fluorescence decay curves, instrumental response function (IRF, grey), bi-exponential (DCM) and tri-exponential (2DCM, 3DCM) curve fits (black), and weighted residuals of DCM (pink), 2DCM (blue) and 3DCM(orange) in THF, before and after irradiation at 485 nm (PSS485). exc = 400 nm, em = 590 nm.

  [a] The global  2 R corresponding to the global analysis of the full series of decay curves was calculated at 1.08, and error bars were estimated based on a 10% tolerance on the global  2 R.[b] Irradiated samples at irr = 485 nm.

Figure 6 .

 6 Figure 6. (Top) Fluorescence anisotropy of DCM (pink), 2DCM (blue) and 3DCM (orange) in propylene glycol at -45°C, collected as a function of excitation wavelength (full circles) and emission wavelength (open circles). (Bottom) Absorption (full lines) and emission spectra (dotted lines) of DCM (pink), 2DCM (blue) and 3DCM (orange) in propylene glycol at -45°C.

Figure 7 .

 7 Figure 7. (a) (EZ)-2DCM and (b) (EE)-2DCM, optimized by the ONIOM method, and used as initial structures for the MD simulations. 𝜇 𝐷 ⃗⃗⃗⃗ refers the donor transition dipole moment vector, 𝜇 𝐴 ⃗⃗⃗⃗ to the acceptor transition dipole moment, 𝜃 𝑇 is the angle between the two vectors, 𝑅 𝐷𝐴 ⃗⃗⃗⃗⃗⃗⃗ is the vector defined between the two mass centers of the DCM units, 𝜃 𝐷 and 𝜃 𝐴 are the angles between 𝑅 𝐷𝐴 ⃗⃗⃗⃗⃗⃗⃗ and 𝜇 𝐷 ⃗⃗⃗⃗ , 𝑅 𝐷𝐴 ⃗⃗⃗⃗⃗⃗⃗ and 𝜇 𝐴 ⃗⃗⃗⃗⃗ , respectively, and 𝜑 is the dihedral angle between the (𝑅 𝐷𝐴 ⃗⃗⃗⃗⃗⃗⃗ , 𝜇 𝐷 ⃗⃗⃗⃗ ) plane and the (𝑅 𝐷𝐴 ⃗⃗⃗⃗⃗⃗⃗ , 𝜇 𝐴 ⃗⃗⃗⃗⃗ ) plane.

  The 200 ns trajectory of EFRET(t) for (EZ)-2DCM is shown in Figure8a, together with the histogram of EFRET. As a result, the EFRET vaues calculated for the (EZ)-2DCM span from 0% to 100%, with an average level of 63.4%, showing that a very wide variety of geometries are visited during the MD simulation, associated to very different FRET efficiencies. The highest probability (largest number of frames) corresponds to EFRET in the 80-100% range, but the statistical distribution has a very flat behaviour, with a large contribution of events where EFRET < 10%. These extreme regions indicate the great conformational variability of the molecule, at the origin of the large dispersion of  2 and/or𝑅 𝐷𝐴 values. Figure 8b,d corresponds to enlarged areas of the EFRET trajectory, their corresponding RDA and  2 being plotted in Figure 8c,e, respectively. During some specific time-periods (58.0-58.5 ns, 183-190 ns), the RDA values are below 1.

  , showing identical trends (two tendency dashed lines in the range 59.2-60.7 ns, where RDA  2).

Figure 8 .

 8 Figure 8. (a) MD trajectory of (EZ)-2DCM hetero-FRET efficiency EFRET (t) during 200 ns, and corresponding histogram of EFRET distribution. (b, d) Enlarged areas of the EFRET (t) trajectory in the time intervals 58-61 ns and 175-190 ns. (c, e) Enlarged areas of the RDA(t) (navy blue) and  2 (t) (yellow) trajectories corresponding to (b, d), respectively. (f) Three selected typical geometries of (EZ)-2DCM, indicated by green vertical bars in (b-e), with green and violet arrows representing the transition dipole moments of the donor (E)-DCM and the acceptor (Z)-DCM units, respectively. Their corresponding FRET efficiencies are marked in the insets.

Figure 9 .

 9 Figure 9. (a) tICA based on 200 ns MD trajectories of (EZ)-2DCM and (b) its tICA-FEL visualized MSM. (c) tICA based on 200 ns MD trajectories of (EE)-2DCM and (d) its tICA-FEL visualized MSM. The κ-means clustering method generated 200 microstates that are lumped into 6 metastable states by the PCCA+ algorithm. The metastable states trajectories are clustered to extract the major conformations in six colors, which correspond to the labels in the transition pathway maps. The size of the labels and the arrows in (b, d) correspond to their proportional coarse-grained fluxes. The percentages of transition pathways are listed above each MSM in (b) and (d).

Table 1 . Photophysical properties of DCM, 2DCM and 3DCM in THF: maximum absorption

 1 and emission wavelengths abs and em, fluorescence quantum yields F, molar absorption coefficients at 457 nm (457nm).

		abs /nm	em /nm	F [a]	(457nm) /L mol -1 cm -1
	DCM	457	590	0.25 ± 0.03	48700 ± 600
	2DCM	457	588	0.21 ± 0.02	88400 ± 1800
	3DCM	457	590	0.21 ± 0.02	128300 ± 1800

Table 2 .

 2 Time

		1 (a1, f1)	2 (a2, f2)	3 (a3, f3)	R Local  2	[a]
	DCM	0.250.08	0.840.03	-	1.02
		(0.11, 0.04)	(0.89, 0.96)
	DCM irr. [b]	0.250.08	0.840.03	-	1.01
		(0.11, 0.03)	(0.89, 0.97)
	2DCM	0.250.08	0.840.03	1.900.50	1.12
		(0.23, 0.08)	(0.74, 0.84)	(0.03, 0.08)
	2DCM irr. [b]	0.250.08	0.840.03	1.900.50	1.07
		(0.32, 0.12)	(0.65, 0.78)	(0.04, 0.10)
	3DCM	0.250.08	0.840.03	1.900.50	1.12
		(0.27, 0.09)	(0.66, 0.73)	(0.07, 0.18)
	3DCM irr. [b]	0.250.08	0.840.03	1.900.50	1.13
		(0.39, 0.15)	(0.55, 0.69)	(0.06, 0.16)

-resolved fluorescence parameters of DCM, 2DCM and 3DCM in THF, before and after irradiation at 485 nm.

Table 3 .

 3 Average

		energy transfer efficiency value (EFRET) classified as a
	function of the interchromophoric distance (RDA), extracted from (EZ)-2DCM
	and (EE)-2DCM MD simulation trajectories.
			average EFRET
		RDA  1	1 < RDA < 2	RDA  2
	(EZ)-2DCM	91.5%	64.7%	23.2%
	(EE)-2DCM	98.5%	92.3%	61.5%

Acknowledgements

We gratefully thank Arnaud Brosseau for his help in running the time-resolved fluorescence instrument. This work was performed by using HPC resources from the "Mésocentre" computing

(http://mesocentre.centralesupelec.fr/). Funding from the Agence Nationale de la Recherche (ANR-17-CE07-0056-01) is acknowledged.


Madelyn Rojas

Marta Avalos

Benjamin Contrand
email: benjamin.contrand@u-bordeaux.fr

Marion Dupuy
email: m.dupuy@calyxis.fr

Catherine Sztal-Kutas

Ludivine Orriols
email: orriolsludi@hotmail.com

Emmanuel Lagarde
email: emmanuel.lagarde@u-bordeaux.fr

Rojas Castro
email: rojas-castro@u-bordeaux.fr

Madelyn Yiseth

Lagarde

Effects of home environmental, behavioural and domestic activities on the risk of home injuries in French adults: Results from a prospective study

Keywords: home, environmental factors, cohort study, web-based, mixed methods n = number of responders, HI = number

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

Home is the environment in which most unintentional injuries occur [START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF][START_REF] Runyan | Unintentional injuries in the home in the United States: Part I: Mortality[END_REF]. Home injuries (HIs) represent a substantial health burden worldwide, approximately one-third of the global burden of injuries [START_REF] Haagsma | The global burden of injury: Incidence, mortality, disability-adjusted life years and time trends from the global burden of disease study 2013[END_REF][START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF][START_REF] Who | WHO Housing and health guidelines[END_REF]. In the EU-28, HIs caused an annual average of 85 thousand fatalities, 1.5 million hospital admissions and 9 million emergencies between 2012 and 2014 [START_REF] Eurosafe | Injuries in the European Union. Summary of injury statistics for the years 2012-2014[END_REF]. In the United States, 75% of all preventable injury-related deaths occurred in homes and communities in 2019 (National Safety Council, 2019). This large HI burden and the current interest in the effect of housing conditions on health disparities stresses the need to understand the effect of exposure to home environmental hazards, together with individual vulnerabilities and social determinants, on the risk of experiencing HI [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF][START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF][START_REF] Swope | Housing as a determinant of health equity: A conceptual model[END_REF][START_REF] Who | WHO Housing and health guidelines[END_REF]. Particular attention was paid to environmental barriers related to functional limitations in community-dwelling older adults [START_REF] Ekström | Home and health among different sub-groups of the ageing population: A comparison of two cohorts living in ordinary housing in Sweden[END_REF][START_REF] Granbom | A public health perspective to environmental barriers and accessibility problems for . senior citizens living in ordinary housing[END_REF][START_REF] Tsuchiya | An acute bout of housework activities has beneficial effects on executive function[END_REF].

Several studies have been conducted to identify injury hazards at home [START_REF] Camilloni | A case-control study on risk factors of domestic accidents in an elderly population[END_REF][START_REF] Ferrante | Home injuries in Italy: Patterns of injury and the most exposed people[END_REF][START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF][START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF] and to measure the effectiveness of hazard modification interventions and prevention strategies for the leading causes of home injury (i.e., home fires and burns, poisonings and falls) [START_REF] Camilloni | A case-control study on risk factors of domestic accidents in an elderly population[END_REF][START_REF] Dionyssiotis | Analyzing the problem of falls among older people[END_REF][START_REF] Farchi | Unintentional home injuries reported by an emergency-based surveillance system: Incidence, hospitalisation rate and mortality[END_REF][START_REF] Gillespie | Interventions for preventing falls in elderly people[END_REF][START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF][START_REF] Schaudt | Accidents Caused by Gardening-Trivial or Serious? 5-Year Retrospective Analysis at the University Emergency Department Berne[END_REF]. Literature reviews showed strong evidence for an association between hazards at home and injuries [START_REF] Carter | Environmental hazards in the homes of older people[END_REF]Keall et al., 2008;[START_REF] Who | WHO Housing and health guidelines[END_REF]. The certainty of the evidence is moderate to high for reducing hazards at home and moderate for the efficacy of hazard modification programs [START_REF] Who | WHO Housing and health guidelines[END_REF].

. According to these studies, some confirmed home hazards related to injury risk are the absence of railings on stairways [START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF], the height of rails and the spaces between the stair rails [START_REF] Keskinoglu | Home accidents in the community-dwelling elderly in Izmir, Turkey: How do prevalence and risk factors differ between high and low socioeconomic districts?[END_REF], insufficient lighting [START_REF] Camilloni | A case-control study on risk factors of domestic accidents in an elderly population[END_REF][START_REF] Farchi | Unintentional home injuries reported by an emergency-based surveillance system: Incidence, hospitalisation rate and mortality[END_REF][START_REF] Gillespie | Interventions for preventing falls in elderly people[END_REF], deteriorated stairs [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF], slippery or uneven floors [START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF] (such as vinyl or linoleum [START_REF] Talbot | Falls in young, middle-aged and older community dwelling adults: Perceived cause, environmental factors and injury[END_REF]), steps or uneven surfaces [START_REF] Talbot | Falls in young, middle-aged and older community dwelling adults: Perceived cause, environmental factors and injury[END_REF], loose rugs or carpets [START_REF] Gillespie | Interventions for preventing falls in elderly people[END_REF][START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF], loose electrical wires on the bedroom floor {Healey1994}, unstable furniture and obstructed walkways [START_REF] Gillespie | Interventions for preventing falls in elderly people[END_REF][START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF], lack of window guards at the second level or higher [START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF], tap water that is too hot [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF], lack of working smoke alarms [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF][START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF], electrical hazards and sources of carbon monoxide (CO) [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF], having a garden [START_REF] Ferrante | Epidemiological risk analysis of home injuries in Italy (1999-2006)[END_REF] and having a balcony [START_REF] Ferrante | Epidemiological risk analysis of home injuries in Italy (1999-2006)[END_REF].

In the same sense, the evidence of the effectiveness of home hazard modifications on falls in the elderly is uncertain [START_REF] Carter | Environmental hazards in the homes of older people[END_REF][START_REF] Clemson | Case-control study of hazards in the home and risk of falls and hip fractures[END_REF][START_REF] Gillespie | Interventions for preventing falls in elderly people[END_REF][START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF][START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF][START_REF] Lyons | Modification of the home environment for the reduction of injuries[END_REF][START_REF] Turner | Risk factors associated with unintentional house fire incidents, injuries and deaths in high-income countries: A systematic review[END_REF].

Effectiveness varies, depending on interacting health factors (history of falls, limited mobility, poor balance, cognitive impairment) [START_REF] Ambrose | Risk factors for falls among older adults: A review of the literature[END_REF][START_REF] Clemson | Case-control study of hazards in the home and risk of falls and hip fractures[END_REF][START_REF] Lord | Home environment risk factors for falls in older people and the efficacy of home modifications[END_REF], risk-taking factors, and the intervention methods used. For instance, grab rails/bars and slips/trips in the bathroom were described to adequately reduce falls in older people with reduced physical abilities [START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF][START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF].

Unsafe behaviours and at-risk behaviours in the domestic environment have been less studied. Participation in activities such as do-it-yourself (DIY), gardening and domestic activities have been associated with a higher incidence of HI in several original studies conducted in the general population [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF][START_REF] Schaudt | Accidents Caused by Gardening-Trivial or Serious? 5-Year Retrospective Analysis at the University Emergency Department Berne[END_REF] [START_REF] Yiengprugsawan | Risk factors for injury in a national cohort of 87,134 Thai adults[END_REF]. Individual, behavioural and environmental factors influence higher risk levels. Knowledge of the relationships between home hazards and injury occurrence is still limited, principally due to insufficient data on exposure and potentially confounding factors [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF][START_REF] Keall | Injuries associated with housing conditions in Europe: A burden of disease study based on 2004 injury data[END_REF][START_REF] Who | WHO Housing and health guidelines[END_REF].

Further research is required to identify new risk factors and understand the effect of environmental and behavioural factors, particularly among the most vulnerable and in other groups where studies are scarce [START_REF] Gielen | Unintentional Home Injuries Across the Life Span: Problems and Solutions[END_REF][START_REF] Peeters | A comprehensive assessment of risk factors for falls in middle-aged adults: Coordinated analyses of cohort studies in four countries[END_REF].

The purpose of this study was to assess associations of a wide range of home environmental, behavioural and domestic activities factors on the risk of non-fatal HI in adults of all ages in the MAVIE cohort. The availability of prospective cohort-based data and the access to relevant exposure information (e.g., socioeconomic information, time spent at home, health status, history of injuries) allows us to study the most relevant associations, some of them unexplored, which can be helpful in the design of future interventions.

Materials and methods

Study design

The MAVIE cohort is a web-based prospective cohort study conducted in France, with a longitudinal follow-up of HLIs [START_REF] Rojas | Cohort profile: MAVIE a web-based prospective cohort study of home, leisure, and sports injuries in France[END_REF]. All households in France and French overseas territories were eligible to participate. The recruitment process began in November 2014. The present study analysed the data collected up to December 31, 2019.

Cohort management was entirely online. Participants were recruited through an email invitation sent to their insurees by three mutual insurance companies, press releases, social media, posters, and flyers. A household reference member was in charge of completing a web-based questionnaire for the household. Consenting members of each household were asked to provide individual information. Caregivers were invited to represent and participate on behalf of one older person (to address the foreseeable difficulties with computer use).

The inclusion criteria for participation in the MAVIE cohort were: i) residing in France;

ii) being able to answer the questionnaires in French; iii) having access to and being able to use the Internet (at least the reference member). The baseline sample was defined as the participants aged 15 or over who answered at least one question of the individual inclusion questionnaire. We excluded those who lived in hospitals and retirement or long-term care institutions. We only considered participants whose followup could be confirmed (by injury or non-injury report, questionnaire update, death or study leave request). We also excluded those participants who did not answer the daily schedule questions.

Events notification change in household composition and moves could be reported at any time. Also, every three months, household reference members received an email reminder to report any injury event to any consenting household members during the follow-up. If no injury had occurred, a link in the email allows reporting with a single click.

Home Injuries

This study focused on unintentional home injuries or its premises. We excluded all events involving illnesses or medical symptoms. We excluded events that occurred before or on the same date as the date of consent and those for which information about the type of medical care or the circumstances of occurrence was not reported. Finally, we excluded injuries that occurred during sleeping time, considered unlikely [START_REF] Kopjar | Population-based Study of Unintentional Injuries in the Home[END_REF].

Data included activity and location, mechanisms and type of medical care of the injury event. We defined HIs as severe when emergency care or hospital inpatient care was required.

Individual characteristics

Gender, age, self-perceived physical and mental health, history of previous injury (excluding sports injuries) over the past 12 months, education status, household incomes, living situation, and alcohol consumption were studied at baseline.

Self-perceived mental health and self-perceived physical health were both selfrated using a visual analogue scale (1 = Poor health, 10 = Excellent health). The responses were classified as: excellent health (8 to 10), good health (5 to 7), and poor health (1 to 4). Occupational status groups were unemployed, homemakers, and retirees and students and employees. Responses to education status were assigned to 'educational attainment levels by age group'. We considered a low level of education in high school or vocational studies, or lower for participants aged 20 to 54, and primary studies or lower for other ages. Annual household incomes were assigned to classes according to the French population's percentiles reported in 2015 (low: ≤ 30th percentile, middle: 30th-80th percentile, high: ≥ 80th percentile). The categories of the frequency of alcohol consumption were less than two times a week, and two or more times a week.

Participants were asked to report their 'typical daily schedule' on a weekday and on a weekend day, the time spent at home and in different domestic settings (dining room, bedroom, garden, etc.). The average duration was also reported for domestic work, gardening, and do-it-yourself (DIY) activities. Time spent on each activity was categorized as frequent (over the 75th percentile), occasional or null.

Household characteristics and behaviours

Household level questions focused on the collection of data on general residence characteristics (location of the residence, type of housing, type of dwelling, age of construction, dwelling area), characteristics of the living environment, including habitat characteristics (interior and exterior), presence of domestic animals, safety equipment, heating system, electrical installations, physical hazards, other hazards and risky behaviours.

Statistical analyses

Data were analysed using R version 3.6.1 (The R Foundation for Statistical Computing, Vienna, Austria)

We defined the follow-up period (FP) from the consent date to the date of the last information reported: injury or non-injury report, the questionnaire's update date, the cohort exit date, the death date. We calculated the proportion of time spent awake at home (PTAH) as the proportion of hours on a weekday or weekend day when the person was neither away from home nor sleeping, for each of the declared households considering moves. We considered the time-at-risk as:

Time-at-risk = (PTAH w × No. FPw) + (PTAH wn × No. FPwn), (1)
where PTAH w was the time spent awake at home on a regular weekday, PTAH wn on a regular weekend day, No. FP w the number of weekdays in the follow-up period and No.

FP wn the number of weekend days. We calculated the proportion of time-at-risk as:

% Time-at risk = (Time-at-risk/Follow-up duration) ×100%

(2).

Risk factor analysis

Poisson mixed models were used to fit the number of HIs during the estimated exposure distribution in the person-time at risk for injury. Repeated measures are included for each individual to account for moves. Different follow-up times were assigned .

according to move dates. Nested random effects were included to consider the cluster structure of data in households. All models were fitted using the Template Model Builder R package [START_REF] Brooks | GlmmTMB Balances Speed and Flexibility Among Packages for Zero-inflated Generalized Linear Mixed Modeling[END_REF]. Relative risks (RRs) were calculated conditionally upon the random effects, and the 95% confidence intervals were calculated using the profile method [START_REF] Venzon | A Method for Computing Profile-Likelihood-Based Confidence Intervals[END_REF]. We adopted a complete case approach.

Confounding factors were pre-selected through 'Directed Acyclic Graphs'. We fitted individual crude regression models to main exposures of interest and confounding factors. We conducted a variable selection procedure by steps and blocks of variables.

In step 1, we adjusted each model by age, gender, history of previous injury, selfperceived physical and mental health status in all models, and we selected the most important associations (P adj < 0.10). In step 2, we fitted the models to each of the exposures of interest, adjusting for significant adjustment variables selected in the previous step. In step 3, we fitted a fully adjusted model with the most important associations. Benjamini-Hochberg corrected p-values were computed [START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing[END_REF]. The same methodology was used to construct models stratified by two distinct age groups (15 to 49 years old and 50 years old and over). The attributable fraction (AF) was estimated using the adjusted RR of the fully adjusted models, as suggested by [START_REF] Flegal | Using adjusted relative risks to calculate attributable fractions[END_REF]colleagues, 2006 (Flegal et al., 2006).

Models were validated with appropriate diagnostic tools, ruling out problems of misspecification, such as over/under dispersion and zero inflation.

Results

Between November 2014 and December 2019, 29931 potential participants were registered in the MAVIE Observatory by the reference members. 41% signed the informed consent and responded to baseline questionnaires. Among them, 11420 were 15 years old or more, 9429 were household reference members representing themselves and/or another household member. Sixty-six participants reported living in a retirement institution, and 3524 participants did not provide follow-up data. Another 1684 participants did not provide their daily schedule (Figure 1). The sample size was 6146 participants from 5122 households.

The median follow-up was 4.0 years (Q1=3.6, Q3=4.5) and the máximum 5.1 years. The overall follow-up was 24480 person-years. The estimated loss to follow-up was 13%.

Among the 6146 individuals of the study, 244 left the cohort during the followup. Nineteen of them were reported by the reference member to have died, 16 from illness, and three unknown causes. Thirty-one participants left the study due to changes in household composition. The remaining 191 participants did not report any reasons for leaving the study.

Baseline characteristics

Among the 6146 adults in the analysis, 3213 were female (52%) and 2933 males (48%), their mean age was 53 ± 15, and 62% of the participants were aged 50-74. More than half reported excellent self-perceived physical (54%) and mental (61%) health (Table 1). In previous studies, we showed that MAVIE participants were older and had higher socioeconomic and educational levels than the French population (Rojas Castro et al., 2021). The median percentage of time spent at home awake was 48% (Q1=36%, Q3=58%). Adults older than 50, unemployed, retirees, homemakers, and those who reported gardening, DIY, and domestic activities were those who spent the most time at home.

Most participants lived in a house (69%), mainly in cities or suburban areas, and 69% lived in municipalities of less than 30000 inhabitants. The average age of the construction was 42 ± 63 years, and the dwelling area was 113 ± 54 m 2 . Eight percent of the participants lived in constructions from before 1900. Most households had a personal outdoor space (70%) and a garage, box or garden hut (75%) with potentially dangerous objects, products and tools. More than half of the residences had stairs (58%).

The characteristics of the individuals and households of the study sample and the baseline sample were similar (Table 1). However, dropout was more frequent among participants aged 15 to 49 and among households with low-income levels. The dwellings of the analysis sample were also more recent than those of the baseline sample.

Home Injuries

During follow-up, 738 (12%) participants reported at least one HI and 979 HI in total.

Among these946 occurred while awake. Most of HIs were minor injuries, and only 28% required either hospitalization or emergency care. Falling was the main mechanism of HI (39%), domestic and gardening activities (42%), the most frequent activities, and the garden (25%) the most frequent location.

Home injury risk factors

Among the 58-home environmental, behavioural and domestic activities factors considered (Table S1), the following were significantly associated with a higher risk of HI: storing household products out of their original packaging and using a stool to reach high places. No environmental characteristics of the dwelling were found significantly associated with a higher risk of HI.

After adjustment for confounding factors (Block 1, Table 2), DIY practices (occasional RR = 1.48; 95% CI, 1.12 -1.94 or frequent RR= 1.75; 95% CI, 1.21 -2.53), storing household products out of their original packaging (RR = 1.43; 95% CI, 1.07 -1.92), and using a stool to reach high places (RR = 1.42; 95% CI ,1.10 -1.83) were associated with a higher risk of HI in adults of all ages, with a small to moderate effect (Block 2, Table 2). Among the adjustment variables, a history of previous injury, high household income and living alone were also significantly associated with the risk of HI in all adults (Block 1, Table 2).

In the fully adjusted model (Table 2), DIY and storing household products outside their original packaging remained associated with HI risk with an AF of 12.1% and 6.4%, respectively. The risk contribution of using a stool to reach high places also remains significant (AF=6.9%), although with a weaker association (RR=1.32; 95% CI, 1.02-1.72).

Age stratification

In adults under 50 years of age, we found that none of the factors assessed were significantly associated with the HI risk after adjustment. In adults aged 50 years of age and over, DIY, use of a stool to reach high places and the presence of stairs were significantly associated with HI (Table S2). DIY was the characteristic with the highest AF (11.1%). Among the adjustment variables, a history of previous injury, selfperceived poor mental and physical health, high household income and living alone were also significantly associated with the risk of HI in adults aged 50 years of age and over (Table S2).

.

Discussion

Here, we present the first study to control for exposure times when assessing the association between home environmental, behavioural and domestic activity factors with the risk of non-fatal HIs.

We followed up 6146 cohort participants residing in French households, of whom 12% reported at least one HI over an average of 5.1 years. In adults of all ages, among the 58 variables evaluated, only the storage of household products outside their original packaging is associated with an increased risk of HI. In adults aged 50 and over, DIY and stools used to reach high places were associated with an increased risk of HI. In adults under 50 years of age, we found no association of exposures of interest with HI. A large and unprecedented number of factors of the physical environment were collected in our study. Despite this, we did not detect any association of these factors with the risk of HI. This result suggests that interventions targeting adults of all ages should not preferentially focus on modifying the physical environment. Instead, it suggests that behaviors play an important role in the risk of HI and in particular among adults aged 50 and over.

Changing the physical environment and mapping hazards in the homes of people with reduced mobility continue to be helpful to keep them safely active [START_REF] Carnemolla | Systematic Review: Evidence on home modifications[END_REF]. The effectiveness of environmental modification interventions to reduce falls in older adults with reduced physical capacity is indeed well described [START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF][START_REF] Gillespie | Interventions for preventing falls in older people living in the community[END_REF].

In households where household products are usually stored out of their original packaging, the increase in the HI risk does not seem solely related to the increase in the number of poisonings. Similarly, among people aged 50 and over who reported using stools to reach high places in their homes, the proportion of HI was higher for falls but also for other mechanisms of injury. These factors may be 'markers' of a lack of knowledge, risk underestimation or careless attitudes towards home safety. The high percentages of participants reporting these behaviours (15% store household products out of their original packaging and 22% use stools to reach high places) suggest that awareness campaigns on domestic risks is a relevant topic.

We confirmed the increased risk of HI associated with DIY practice in adults aged 50 and over [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF][START_REF] Yiengprugsawan | Risk factors for injury in a national cohort of 87,134 Thai adults[END_REF]. In our study, the risk of HI was 63% higher among frequent DIYers and 40% higher among occasional DIYers, compared to people who did not report it. DIY was most common among people aged 50-69 years and retired people. Crushing and overexertion were the most frequent HI mechanisms (30.6% and 17.2%). The lack of experience and knowledge, underestimation of risks, overconfidence and cost of services are factors that can increase vulnerability to DIY injuries [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF][START_REF] Verrier | Home and leisure injuries among the French electricity and gas company active employees: Circumstances and short-term consequences[END_REF].

Work at home could be another reason [START_REF] Driscoll | Unintentional fatal injuries arising from unpaid work at home[END_REF], but less probable in adults over-50s.

We detected a slight, non-significant association between gardening activities and the risk of HI. Consistent with an original study conducted in Switzerland [START_REF] Schaudt | Accidents Caused by Gardening-Trivial or Serious? 5-Year Retrospective Analysis at the University Emergency Department Berne[END_REF], gardening-related HI were very frequent but most with minor consequences. The authors of this study highlight the incorrect use of gardening tools as a frequent cause of injuries [START_REF] Schaudt | Accidents Caused by Gardening-Trivial or Serious? 5-Year Retrospective Analysis at the University Emergency Department Berne[END_REF]. We found no association of HI with domestic activities such as cleaning and/or cooking.

Intervention focuses on DIY, gardening or of any other domestic activities should not discourage their practice. Leisure activities that keep people active have beneficial effects on health and wellbeing [START_REF] Han | An analysis of the influence that leisure experiences have on a person's mood state, health and wellbeing[END_REF][START_REF] Jiang | The association between mild cognitive impairment and doing housework[END_REF], one example is the moderate practice of gardening [START_REF] Thompson | Gardening for health: A regular dose of gardening[END_REF]. Interventions may inform about existing risks, improve skills, promote risk self-assessment and selfmanagement, such as the use of protective equipment.

A secondary result of this study, consistent with previous literature [START_REF] Camilloni | A case-control study on risk factors of domestic accidents in an elderly population[END_REF][START_REF] Ferrante | Epidemiological risk analysis of home injuries in Italy (1999-2006)[END_REF] was an increased risk of HI (86% higher) for people aged over-50s living alone. This higher risk may however be related with the involvement of older people in DIY.

An important number of aged people choose DIY to improve their general fitness, to feel satisfied and proud of a job well done, and to give meaning and pleasure to everyday tasks [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF]. However, accumulated health problems and functional impairments increase the risk of many tasks, such as working at heights [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF].

Others, despite their state of health or lack of experience, may be forced to take risks because they live alone, do not have access to appropriate resources and services, or are afraid to use them for fear of financial or even physical abuse. [START_REF] Ashby | Investigating the over-representation of older persons in do-it-yourself home maintenance injury and barriers to prevention[END_REF]. The lack of maintenance would expose the person to other hazards. Accessible alternatives should be a priority when self-regulation is insufficient.

The lack of associations in adults under 50 years of age with any of the potential risk factors assessed in this study may be explained by the adjument on perceived health.

The next step would be to investigate an integrated model that includes possible interactions between health, environmental and behavioural factors.

Limitations and Strengths

Common limitations observed in volunteer-based cohorts and e-cohorts are low response rates, volunteer bias, loss of follow-up, and self-administered questionnaires, leading to selection biases and missing and selective answers. As with other e-cohorts [START_REF] Nittas | Toward a working definition of ecohort studies in health research: Narrative literature review[END_REF], we made continuous efforts to address representativeness, data reliability, under-reporting of the outcome, and loss to follow-up [START_REF] Kristman | Loss to follow-up in cohort studies: How much is too much[END_REF][START_REF] Rojas | Cohort profile: MAVIE a web-based prospective cohort study of home, leisure, and sports injuries in France[END_REF].

We mitigated problems related to self-reported information by using an online survey designed to minimize missing or inaccurate data, removing non-essential questions, improving user-friendliness, using intermittent saving options, and checking the consistency of answers to the different questions. Moreover, we used several strategies to reduce under-reporting of the outcome and loss of follow-up, such as quarterly email reminders and one-click event notifications. We estimated a follow-up rate of 86% [START_REF] Rojas | Cohort profile: MAVIE a web-based prospective cohort study of home, leisure, and sports injuries in France[END_REF], which is higher than the 60-80% range considered acceptable by some authors for a valid study [START_REF] Kristman | Loss to follow-up in cohort studies: How much is too much[END_REF].

Our sample is not representative of the French population. Young adults and people from low socioeconomic groups and low levels of educational attainment were underrepresented [START_REF] Rojas | Cohort profile: MAVIE a web-based prospective cohort study of home, leisure, and sports injuries in France[END_REF]. Dwellings with security problems are also underrepresented. In a group of volunteers motivated to participate in a health study, socioeconomic and lifestyle differences between exposed and unexposed individuals would be less critical than in a representative sample of the general population [START_REF] Nohr | Commentary: Epidemiologists have debated representativeness for more than 40 years has the time come to move on[END_REF][START_REF] Richiardi | Commentary: Representativeness is usually not necessary and often should be avoided[END_REF][START_REF] Rothman | Why representativeness should be avoided[END_REF]. Even if exposure heterogeneity was not captured, the greater homogeneity of the control factors might have increased the statistical power to detect main effects. Homogeneity may be desired when the multifactorial aspect is analysed.

We cannot rule out the existence of misclassification, especially for characteristics perceived as of minor importance. We think that the involvement of a reference person to communicate household information might increase the reliability of the data. The high follow-up rate also suggests that participants are motivated, increasing the reliability of the responses.

This study design does not allow us to disentangle the effects of risk associated with the activity itself and the risk of the tool or the product. We do not have information on the individual use of those products. We measured exposures at baseline; modifications in the environment, behaviours or the practice of activities were not assessed over time. Moreover, information about individual risk-taking and risk awareness was also not available. The sample size and the number of events prevented analyses in other population groups of interest, such as children and people with disabilities and different injury mechanisms.

Conclusion

This study suggests that improving risk perception and self-regulation may be a key

which was not certified by peer review)

  ., . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (

	medRxiv preprint	doi:	; https://doi.org/10.1101/2022.07.18.22277761	this version posted July 19, 2022.	The copyright holder for this preprint
	2019;
					5

  target for the development of effective interventions to reduce the number of HI.

	High	4949	(47)	3206	(50)
	missing	1548		682
	Location of residence
	(municipality)
	Rural (less than 2,000 inhab.)	2486	(22)	1508	(22)
	2,000 -5,000 inhab.	2092	(18)	1228	(18)
	5,000 -30,000 inhab.	3303	(29)	1967	(29)
	30,000 -100,000 inhab.	1731	(15)	1074	(16)
	More than 100,000 inhab.	1764	(16)	1106	(16)
	missing	708		227
	Type of housing
	Houses dispersed, outside the urban
		2172	(19)	1252	(18)
	area
	Houses in a housing estate, suburban
		5464	(47)	3388	(48)
	area or city
	Buildings in a city or large complex	2158	(19)	1308	(19)
	Interventions aimed at making DIY safer are likely to reduce HI. Awareness-raising Buildings other than a city or large
	complex campaigns on HI are still needed and enable them to manage them themselves. 347 (3) 187	(3)
	Mixed housing: both buildings and
		1470	(13)	864	(12)
	houses
	missing	473		111
	Type of dwelling
	House	7919	(68)	4803	(69)
	Apartment	3634	(31)	2164	(31)
	Other	85	(1)	40	(1)
	missing	446		103
	Age of construction (years)	Mean	S. D	Mean	S. D
		58	61	42	63
	Dwelling area (m 2 )	Mean	S. D	Mean	S. D
		112	54	113	54

Table 2 .

 2 Home environmental, behavioural and domestic activities factors associated with the incidence of HI, relative to the time at risk at home, in adults of the MAVIE cohort (adjusted and fully adjusted models)

		Adjusted Models*			Fully adjusted model †
		(n = 4873, HI = 648)			(n = 4579, HI = 609)
					n		AF
		n (%)	RR (95% CI)	P c		RR (95% CI)	P c
					(%)		(%)
	Block 1: Adjustment	(model including only the
	variables	adjustment variables)
	Gender
	Female (vs. male)	(53) 0.98 (0.77 -1.25) 0.875 (53)	1.18 (0.91 -1.54) 0.306	7.7
	Age (years)
	15-29 (vs. 30-49)	(10) 1.24 (0.77 -2.00) 0.829 (10)	1.29 (0.80 -2.08) 0.541	1.6
	50-74	(58) 0.96 (0.72 -1.28)	-(59)	0.91 (0.68 -1.21)	--6.6
	75+	(4) 0.91 (0.50 -1.68)	-	(4)	0.94 (0.52 -1.70)	--0.3
	History of previous
	injury (12 last
	months)
	Yes (vs. no)	(9) 1

.64 (1.14 -2.34) 0.021 (9) 1.60 (1.12 -2.27) 0.029

	Education level
							12.
	High (vs. low)	(92) 1.25 (0.78 -2.01) 0.462 (92)	1.15 (0.72 -1.82) 0.560
							1
	Household income
	level					-
							15.
	High (vs. middle)	(49) 1.40 (1.08 -1.82) 0.004 (49)	1.37 (1.06 -1.78) 0.006
							1
	Low	(12) 0.61 (0.40 -0.95)	-(12)	0.61 (0.39 -0.93)	--5.4
	Living alone
							11.
	Yes (vs. no)	(22) 1.77 (1.31 -2.39) 0.002 (22)	1.79 (1.33 -2.41) 0.001
							2
	Frequency of alcohol
	consumption
	2 or more times a week
		(40) 1.23 (0.96 -1.57) 0.213 (41)	1.21 (0.95 -1.54) 0.223	8.2
	(vs. <2 times a week)
	Block 2: Exposure	(individual models adjusted for
	variables	variables of the block 1)
	DIY	4360
	Occasional (vs. never)	(30) 1					4.9
	Self-perceived
	physical health
	Poor (1 to 4) vs.
		(8) 1.07 (0.83 -1.39) 0.462	(8)	1.38 (0.90 -2.13) 0.412	2.5
	Excellent (8 to 10)
	Good (5 to 7)	(39) 1.37 (0.89 -2.12)		(38)	1.07 (0.83 -1.38)	-	3.0
	Self-perceived mental
	health
	Poor (1 to 4) vs.
		(6)	0.423	(6)	1.50 (0.93 -2.40) 0.306	0.5
	Excellent (8 to 10)
	Good (5 to 7)	(33) 1.01 (0.78 -1.32)	-(33)	1.02 (0.78 -1.32)	-	2.8

.48 (1.12 -1.94) 0.007 (31) 1.48 (1.13 -1.95) 0.012

							12.
							1
	Frequent	(12) 1.75 (1.21 -2.53)	-	(12)	1.75 (1.21 -2.53)	-	7.5
	Storing cleaning
	products out of their	4796
	original packaging
	Yes (vs. no)	(15) 1.53 (1.15 -2.04) 0.007	(15)	1.43 (1.07 -1.92) 0.039 6.4
	Using a stool to reach
		4764
	high places
	Yes (vs. no)	(23) 1.42 (1.10 -1.83) 0.009	(24)	1.32 (1.02 -1.72) 0.069 6.9

Figures

Figure 1.

Flow diagram for the selection of study participants