# Dietary isothiocyanates inhibit the oxidative activity of salivary aldehyde dehydrogenase ALDH3A1 and modulate aroma release

Valentin Boichot, Jeanne Chaloyard, Karine Gourrat, Jade Moreno, Jean-Michel Saliou, Jean-Marie Heydel, Francis Canon, Fabrice Neiers, Mathieu Schwartz

► To cite this version:

Valentin Boichot, Jeanne Chaloyard, Karine Gourrat, Jade Moreno, Jean-Michel Saliou, et al. Dietary isothiocyanates inhibit the oxidative activity of salivary aldehyde dehydrogenase ALDH3A1 and modulate aroma release. Food Chemistry, 2026, 508 (Part A), pp.148324. 10.1016/j.foodchem.2026.148324. hal-05514870

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# Dietary isothiocyanates inhibit the oxidative activity of salivary aldehyde dehydrogenase ALDH3A1 and modulate aroma release

Valentin Boichot<sup>a</sup>, Jeanne Chaloyard<sup>b</sup>, Karine Gourrat<sup>a,c</sup>, Jade Moreno<sup>a,e</sup>,
Jean-Michel Saliou<sup>d</sup>, Jean-Marie Heydel<sup>b</sup>, Francis Canon<sup>a</sup>, Fabrice Neiers<sup>b,*</sup>,
Mathieu Schwartz<sup>a,e,1,**</sup>

## ARTICLE INFO

### Keywords:

Isothiocyanate

ALDH3A1

Salivary enzymatic activity

Octanal

Aroma release

Aldehyde dehydrogenase

## ABSTRACT

This study investigates the inhibitory effects of dietary isothiocyanates (ITCs) on the enzymatic activity of salivary aldehyde dehydrogenase ALDH3A1, a key enzyme involved in the metabolism of odorant aldehydes and abundantly expressed in the oronasal sphere. Using a combination of enzymology, X-ray crystallography and mass spectrometry, we demonstrate that ITCs significantly inhibit ALDH3A1 activity both *in vitro* and *ex vivo* in saliva. Our findings reveal that allyl-isothiocyanate forms a covalent adduct with the ALDH3A1 catalytic cysteine residue (Cys243) leading to an irreversible inhibition. This inhibition disrupts the metabolic conversion of aldehydes in saliva, thereby impacting aroma release. GC-MS analysis confirmed that ITC-mediated inhibition of salivary ALDH activity modulates the concentration of odorant metabolites in both liquid and gas phases. These results highlight the significant influence of dietary ITCs on salivary enzymatic activity and potential implications for aroma release and flavor modulation in the oral cavity.

## 1. Introduction

A diet rich in vegetables promotes good health and can help prevent a wide range of diseases. This protective effect is primarily attributed to the abundant nutrients, dietary fibers and phytochemicals found in vegetables (Boeing et al., 2012). Among these phytochemicals, isothiocyanates (ITCs) are sulfur-containing phytochemicals present in cruciferous vegetables, particularly in the Brassicaceae family, which includes Brussels sprouts, cabbage, cauliflower, radish, and kale (Olayanju et al., 2024). Many investigations have shown that different isothiocyanates exhibit a wide range of biological activities, including anticancer, anti-inflammatory, and antioxidant effects (Olayanju et al., 2024; Yagishita et al., 2019). ITCs are primarily formed in plants from glucosinolate precursors through the action of an enzyme called myrosinase. This reaction occurs when plant tissues undergo stress, such as biting, slicing, or chewing, leading to the release of myrosinase (Oliviero

et al., 2018). The ITCs produced in this process are believed to protect the plant from herbivores by generating a bitter and pungent taste (Bones & Rossiter, 1996). While glucosinolates themselves are relatively unreactive, ITCs can readily interact with thiol and amine groups in peptides and proteins (Brown & Hampton, 2011). Due to their high reactivity, ITCs can chemically modify certain enzymes, including xenobiotic-metabolizing enzymes (XMEs), potentially leading to their inhibition (Kumari et al., 2016; Nakajima et al., 2001).

XMEs play a crucial role in the detoxification of xenobiotics from our daily environment, including odorants, pollutants, and food-derived molecules. These enzymes metabolize xenobiotics to increase their water solubility, facilitating their conjugation with hydrophilic molecules and then their elimination. Evidence suggests a protective role of oronasal XMEs in metabolizing and neutralizing xenobiotics entering the oral and nasal cavities (Heydel et al., 2019; Poirier et al., 2024). Additionally, oronasal XMEs can process flavor compounds, thereby

<footnote><sup>a</sup> Université Bourgogne Europe, INRAE, Institut Agro, CNRS, UMR CSGA, F-21000 Dijon, France</footnote>

<footnote><sup>b</sup> Université Bourgogne Europe, CNRS, Laboratoire Interdisciplinaire Carnot de Bourgogne ICB, UMR 6303, F-21000 Dijon, France</footnote>

<footnote><sup>c</sup> INRAE PROBE Research Infrastructure, Chemosens Facility, F-21000 Dijon, France</footnote>

<footnote><sup>d</sup> Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UAR CNRS 2014 - US Inserm 41 - PLBS, Lille, France</footnote>

<footnote><sup>e</sup> Université Bourgogne Europe, INRAE, Institut Agro, UMR PAM, F-21000 Dijon, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: fabrice.neiers@u-bourgogne.fr (F. Neiers), mathieu.schwartz@inrae.fr (M. Schwartz).</footnote>

<footnote n="1">¹ Present address: Université Bourgogne Europe, INRAE, Institut Agro, UMR PAM, 21000 Dijon, France</footnote>
altering the quantity and quality of flavor molecules that activate chemoreceptors (Boichot et al., 2022; Robert-Hazotte et al., 2022; Schwartz et al., 2021). Flavor encompasses all sensory perceptions experienced while eating and plays a pivotal role in human dietary preferences, significantly impacting health and overall well-being (Nordin et al., 2011). It is categorized into three sensory modalities: olfaction, taste, and trigeminal sensations. XMEs are present in oronasal fluids (saliva, olfactory mucus), epithelia, and sensory neurons (Heydel et al., 2017; Neiers et al., 2021; Schwartz, Neiers, et al., 2020). These enzymes can interact with or metabolize aroma and taste compounds near chemosensory receptors, thereby modulating chemosensory signaling (Robert-Hazotte et al., 2022; Schwartz et al., 2022). In 2021, our group provided a comprehensive overview of current knowledge on the ability of oral XMEs to react with aroma compounds, generating new metabolites (Schwartz et al., 2021). Recent studies have further highlighted the capacity of certain XMEs such as glutathione transferases (GSTs), to interact with a range of aroma compounds (Hjeij et al., 2024; Robert-Hazotte et al., 2019; Schwartz, Menetrier, et al., 2020) and taste compounds (Schwartz et al., 2022), leading to signal termination (Legendre et al., 2014). Additionally, other enzymes, such as aldehyde dehydrogenases (ALDHs), can transform aroma compounds through oxidoreductive reactions, resulting in new metabolites (Boichot et al., 2023).

ALDH enzymes belong to phase I XMEs dedicated to the functionalization of the xenobiotic molecules and are highly represented in the human nasal and buccal cavities, as well as in olfactory mucus and cilia, where they metabolize odorant molecules such as aldehydes (Boichot et al., 2023; Giebultowicz et al., 2009; Kuhlmann et al., 2014). Aldehydes are widely present in food, with over 550 identified food products containing flavoring aldehydes as natural constituents or additives. These include various aroma compounds such as vanillin (contained in vanilla), octanal (contained in grape, lemon and peel oil), and cinnamaldehyde (contained in cinnamon), which are among the most frequently encountered (Nijssen, 1996). Some aldehydes are known to be metabolized by NAD(P)(H)-dependent oxidoreductases present in saliva and oral mucosa cells, making them less persistent compared to non-metabolized aroma compounds (Munoz-Gonzalez et al., 2018, Munoz-Gonzalez et al., 2021). Among these oxidoreductases, aldehyde dehydrogenase family 3 member A1 (ALDH3A1) has been identified as the major salivary ALDH (Giebultowicz et al., 2009), efficiently metabolizing both aliphatic aldehydes (typically around eight carbons) and aromatic aldehydes (Boichot et al., 2023). ALDH3A1 employs a NAD-dependent mechanism using a catalytic cysteine residue Cys243 to metabolize aldehydes into their corresponding carboxylic acids (Khanna et al., 2011). While ITCs are known to react with nucleophilic cysteine residues, their inhibitory effect on ALDH3A1 activity has not yet been explored. Given the typical concentrations of isothiocyanates in Brassica vegetables such as red cabbage, broccoli, or watercress (up to 10 µg of ITCs per 100 g of fresh tissue; Fusari et al., 2020), the transient oral exposure during mastication can be estimated to reach tens to a few hundred micromolar of isothiocyanates released into the saliva. Such exposure levels may be sufficient to transiently inhibit salivary enzymes.

In this study, we explored the impact of dietary ITCs on the catalytic activity of human ALDH3A1, the predominant salivary ALDH. ALDH3A1 was selected for this study as it represents the major ALDH isoform expressed in the oronasal tissues and fluids (Boichot et al., 2023; Giebultowicz et al., 2009), where its capacity to oxidize volatile aldehydes suggests a potential role in modulating aroma availability and perception. We provide evidence of the inhibitory effect of eight ITCs on the activity of recombinantly produced ALDH3A1 using a combination of biochemical and enzymatic techniques. Furthermore, we elucidated the detailed mechanism of ALDH3A1 inhibition by allyl- isothiocyanate at the atomic level using mass spectrometry and structural biology analysis. Finally, a gas chromatography-mass spectrometry (GC-MS) study revealed inhibition of aroma compound metabolism in saliva, highlighting the potential targeting of other salivary enzymes

by isothiocyanates and their involvement in the biotransformation of odorant aldehydes. This study provides the first evidence that dietary isothiocyanates can directly inhibit the salivary enzyme ALDH3A1, a key player in the metabolism of aroma-active aldehydes in the oral cavity. By demonstrating a covalent inhibition mechanism and revealing how individual variability in salivary composition may modulate this effect, our work uncovers a previously unexplored biochemical link between diet-derived electrophiles and oral enzyme activity. These findings open new perspectives for understanding how dietary compounds influence flavor perception and interindividual differences in sensory responses.

## 2. Material and methods

### 2.1. Chemicals

Unless otherwise specified, all chemicals were purchased at the highest available purity from Sigma-Aldrich (St. Louis, MO, USA).

### 2.2. Production and purification of recombinant ALDH3A1

Human ALDH3A1 was produced and purified as previously described (Boichot et al., 2023). Briefly, the DNA sequence encoding the *Homo sapiens* ALDH3A1 (UniProt code P30838) was optimized for expression in *Escherichia coli* and subcloned into a pET22b vector between the *NdeI* and *SacI* restriction sites. A 6-histidine tag was added at the N-terminal extremity of ALDH3A1 to facilitate protein purification. *E. coli* BL21(DE3) Star were transformed with the plasmid and spread on LB-agar plates supplemented with ampicillin (100 µg/mL). The transformed bacteria were grown in LB medium (supplemented with ampicillin at the same concentration), and overexpression of the recombinant ALDH3A1 was triggered by the addition of 0.1 mM IPTG. Twenty-four hours after induction, bacterial growth was stopped by centrifugation, suspended in Tris buffer (50 mM) pH 8.0, sucrose (250 mM), and lysed at 4 °C by mechanical disruption using two cycles of sonication. After centrifugation at 20,000g for 45 min at 4 °C, ALDH3A1 was purified from the supernatant using two successive chromatography steps. Firstly, a His-trap (Cytiva, Marlborough, USA) column connected to an ÄKTA purification system was used. Partially pure ALDH3A1 was eluted using a gradient of imidazole (500 mM) in a HEPES buffer, pH 7.8 (20 mM) containing NaCl (300 mM), and DTT (100 µM). Secondly, a mono-Q column (Cytiva, Marlborough, USA) was used, and elution was performed using a gradient of NaCl (300 mM) in a HEPES buffer (10 mM), pH 7.8. After each chromatography step, eluates were dialyzed in HEPES (10 mM), NaCl (50 mM), pH 7.8. Subsequently, ALDH3A1 fractions sufficiently pure for enzymatic activity tests and crystallization assays were pooled, concentrated to 1 g.L⁻¹ using a concentrator with a 10 kDa cut-off (Thermo Fisher Scientific, Waltham, USA), and stored at -20 °C.

### 2.3. Enzymatic inhibition assays

The ability of ITCs to modify the efficiency of substrate oxidation of ALDH3A1 was tested using a screening strategy. Enzymatic activity of recombinant ALDH3A1 was determined on a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan) at 25 °C by measuring absorbance at 340 nm corresponding to the NADH absorbance wavelength. Enzymatic reactions were performed in 1 mL quartz cuvette filled with 1 mL of a mix containing 100 mM KPi buffer pH 7.0, 1 mM of NAD, 70 nM of recombinant ALDH3A1, one ITC among allyl-ITC, benzyl-ITC, butyl-ITC, erucin, hexyl-ITC, iberin, 2-phenethyl-ITC and propyl-ITC at 10 or 100 µM. The enzymatic reactions were triggered by adding a saturated concentration of octanal at 100 µM after two different incubation times, 3 or 15 min, and measured during one minute to obtain the initial reaction rates. Each experiment was repeated independently three times. The inhibition percentages of enzymatic activity were calculated using
the following eq. ($1 - (\frac{A_1}{A_0}) \times 100$) where A₁ is the absorbance measured at 340 nm in the sample with ITC and A₀ is the absorbance measured at 340 nm in the sample without ITC. The inhibition percentage is an average from 3 independent measurements. To determine the inhibition constants (Kᵢ) of B-ITC, a similar strategy was employed using three concentrations of B-ITC (10, 50, and 100 µM) without pre-incubation time with saturating concentrations of octanal (20 µM) and NAD (1 mM). The kinetic parameters (Vₘ and Kₘ) were derived from the fitting of the initial velocities to the Michaelis-Menten equation using the SigmaPlot software (version 14.0.0.124, Systat Software, Inc). Double-inverse Lineweaver-Burk plots allowed to identify the competitive inhibition mode. The inhibition constants were calculated accordingly using the equation corresponding to the inhibition mode, here the competitive inhibition: $V_0 = \frac{V_{max} [S]}{K_m (1 + \frac{U}{K_f}) + [S]}$.

## 2.4. X-ray crystallography

Crystallogenesis experiments were conducted using freshly purified ALDH3A1. Prior to crystallization assays, enzymes were dialyzed in 10 mM pH 7.8 HEPES buffer. Crystallization trials were manually performed at 20 °C using the sitting drop vapor diffusion method. ALDH3A1 (2 mg.mL⁻¹) was crystallized by combining 1 µL of protein with 1 µL of a solution consisting of 18% PEG 3350 in 0.1 M potassium acetate at pH 7.5. For the generation of ALDH3A1:allyl-ITC complexes, crystals were soaked in the mother liquor supplemented with 10 mM allyl-ITC. Cryoprotection was achieved by introducing 20% glycerol into the drops containing the crystals. Subsequently, the crystals were rapidly frozen in liquid nitrogen prior to synchrotron data collection (ESRF beamline FIP2-BM07). ALDH3A1:A-ITC crystals diffracted to 2.05 Å. The dataset was indexed and integrated with XDS (Kabsch, 2010) and scaled with aimless (Winn et al., 2011). The structure was solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010) using the coordinates of apo ALDH3A1 (pdb code 3SZA). The structure was manually adjusted with COOT (Emsley and Cowtan 2004) and refined with PHENIX (Adams et al., 2010). Inspection of electron density maps allowed the identification and modeling of allyl-isothiocyanate bound to Cys243. Validation was carried out with MolProbity (Chen et al., 2010). Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 9IG2.

## 2.5. Protein mass spectrometry analysis

Freshly produced recombinant ALDH3A1 (10 µM) was incubated with or without allyl-isothiocyanate at 500 µM during one hour at room temperature. Dialysis was carried out three times in HEPES buffer pH 7.8 to remove excess of inhibitor. Samples were then sent in dry ice for tandem mass spectrometry analysis. Protein samples were loaded on SDS-PAGE to perform one gel slice trypsin digestion for each sample. Peptides were extracted with 0.1% formic acid in acetonitrile, evaporated to reduce volume at 8 µL and injected on an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific). Peptides were automatically fractionated onto a commercial C18 reversed-phase column (75 µm × 150 mm, 2-µm particle, PepMap100 RSLC column, Thermo Fisher Scientific (Waltham, USA), temperature 35 °C). Trapping was performed for 4 min at 5 µL/min with solvent A (98% H₂O, 2% acetonitrile and 0.1% formic acid). Elution was performed using two solvents, A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile), at a flow rate of 300 nL/min. Gradient separation was 2 min at 5% B, 19 min from 5% to 40% B, 2 min from 40% to 80% B, and maintained for 14 min. The column was equilibrated for 6 min with 3% buffer B prior to the next sample analysis. The eluted peptides from the C18 column were

analyzed by Q-Exactive instruments (Thermo Fisher Scientific, Waltham, USA). The electrospray voltage was 1.9 kV, and the capillary temperature was 275 °C. Full mass spectrometry (MS) scans were acquired in the Orbitrap mass analyzer over the m/z 300–1200 range with a resolution of 35,000 (m/z 200). The target value was 5.00E+05. The three most intense peaks with charge states between 2 and 4 were fragmented in the high-energy collisional dissociation cell (HCD) with a normalized collision energy of 35%, and the tandem mass spectrum was acquired using an Orbitrap mass analyzer with a resolution of 17,500 at m/z 200. The target value was 5.00E+04. The ion selection threshold was 5.0E+03 counts, and the maximum allowed ion accumulation times were 250 ms for full MS scans and 150 ms for tandem mass spectra. The dynamic exclusion was set to 7 s.

Raw data collected during nano LC-MS/MS analyses were processed with Proteome Discoverer 2.5 (Thermo Fisher Scientific) using the search engine Mascot (version 2.4.0, Matrix Science, London, UK) installed on a local server. Searches were performed with a tolerance on mass measurement of 0.02 Da for precursor and 10 ppm for fragment ions, against a composite target-decoy database built with a human Swissprot database fused with recombinant trypsin and a list of classical contaminants. Cysteine carbamidomethylation, methionine oxidation, protein N-terminal acetylation, cysteine propionamidation and allyl-isothiocyanate modification were searched as variable modifications. Up to one missed trypsin cleavage was allowed. For each sample, peptides were filtered out according to the cutoff set for peptides hits with ion score > 20.

## 2.6. Measurement of salivary aldehyde dehydrogenase (ALDH) activity

The salivary ALDH activity was assessed using the same methodology as above, with the only difference being the substitution of recombinant ALDH3A1 with 200 µL of freshly collected saliva. Saliva samples were collected in the mornings of experimental days from three distinct fasting donors, all free from taste disorders (male subjects aged 23–29 years). A buffer solution consisting of KPI at pH 7.0, EDTA, and DTT was added to the saliva samples in an equal volume, resulting in final concentrations of 100 mM KPI, 0.5 mM EDTA, and 0.5 mM DTT. The salivary ALDH activity was measured in a 1 mL quartz cuvette filled with a mixture containing 100 mM KPI buffer at pH 7.0; 1 mM NAD and 200 µL of saliva, prepared as previously explained. To initiate the enzymatic reaction, 100 µM of octanal was added to the mixture. Absorbance measurements at 340 nm were taken during the incubation at 37 °C, at time points including 15, 30, 60, 120, and 180 min. Before each measurement, the solutions were centrifuged at 10,000g at 4 °C for 10 min to separate the pellet from the supernatant, allowing for the monitoring of NADH accumulation in the supernatant. The pellet was not discarded to retain the entire saliva, including potential epithelial and microbial cells, which can participate in biotransformation reactions as previously observed (Neiers et al., 2022). The pellet was then resuspended in the supernatant through vortexing and the mixture was incubated again at 37 °C until the next measurement. To evaluate the influence of benzyl-ITC on salivary activity, 100 µM of benzyl-ITC was introduced into the initial mixture for one condition, including 15 min of preincubation before the addition of octanal. Absorbance results for each time point were compared with and without ITC. Negative controls were established either by replacing NAD with water, substituting fresh saliva with saliva heated to 90 °C for 10 min, or replacing octanal (diluted in methanol) with pure methanol.

## 2.7. HS-SPME-GC/MS Headspace solid-phase microextraction Gas Chromatography–Mass Spectrometry Analysis

HS-SPME-GC/MS analyses were performed to investigate the metabolization products resulting from the interaction of octanal with ALDH3A1. Samples were prepared in 10 mL vials containing 1 mL of a reaction mixture composed of 250 nM ALDH3A1, 500 µM octanal, 100
mM KPi buffer, pH 7.0, and 1 mM NAD. The metabolism of octanal by salivary enzymes was assessed using the same protocol, replacing recombinant ALDH3A1 with 500 µL of freshly collected morning saliva. Saliva was obtained from three individuals, and each condition was tested in triplicate for each donor. The impact of cofactor addition (NAD, NADH) and ITCs (A-ITC, B-ITC) on octanal metabolism by salivary enzymes was also examined. Results for NADH addition are presented in the supplemental data. Controls were prepared by replacing ALDH3A1 or saliva with water, and blanks of pure octanal and pure saliva were analyzed under identical conditions to rule out potential artifacts. The effect of ITCs on enzymatic activity was assessed by incorporating 100 µM A-ITC or B-ITC into the reaction mix, with octanal added 15 min later. Vials were sealed with silicone septa (Supelco Sigma-Aldrich, Bellefonte, USA) and incubated at 37 °C for 10 min. Volatile compounds were extracted using a 2-cm triple-phase divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber (Supelco Sigma-Aldrich, Bellefonte, USA) for 10 min at the same temperature. The fiber was then desorbed for 3 min in the injector of a 6890 A GC (splitless mode) maintained at 240 °C. Separation of volatile compounds was achieved using a DB-WAX polar capillary column (30 m × 0.25 mm i.d., 0.5 µm film thickness) from Agilent (J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas at a flow rate of 1.2 mL/min (linear velocity: 40 cm/s). The oven temperature was programmed to increase from 40 to 240 °C at a rate of 6 °C/min.

For the MS system (5973 N quadrupole MSD, Agilent Technologies, Santa Clara, USA), the transfer line, quadrupole, and ion source temperatures were maintained at 250, 150, and 230 °C, respectively. Electron ionization (EI) spectra were acquired at 70 eV with a scan rate of 4 scans per second, covering an *m/z* range of 29–350 uma. Compound identification was based on retention indices (RIs) and mass spectra. RI values were calculated using the Van den Dool and Kratz formula from the retention times of n-alkanes (C6–C30) analyzed on the same column. These values were then compared with those reported in the literature. Mass spectra were matched against the NIST, WILEY, and INRAMASS (internal database of standard compounds) libraries. Additionally, RI values and mass spectra of identified metabolites were verified against pure reference compounds (octanal, octanol, and octanoic acid) using the same analytical procedure to confirm their identification. To investigate the influence of ITCs on metabolite production by recombinant ALDH3A1 or saliva, four experimental conditions were tested. The first condition served as a negative control, consisting of a mixture of 100 mM KPi buffer, pH 7.0, 500 µM octanal, and water. The second condition included the same mixture, supplemented with either 250 nM recombinant ALDH3A1 or 500 µL of fresh saliva, along with 1 mM NAD. In the third condition, 100 µM A-ITC was added to the composition of condition 2. In the fourth condition, 100 µM B-ITC was added to the composition of condition 2.

## 2.8. LLE-GC/MS

Liquid-Liquid Extractions (LLE) were performed to analyze the liquid phase within the vials. After 10 min of incubation followed by 10 min of adsorption period, 500 µL of dichloromethane was added to stop enzymatic reactions, and the vials were stored at –20 °C. Storage time did not affect the composition of the liquid phase. The vials were then vortexed for homogenization and centrifuged at 15,000g and 4 °C for 15 min to separate the organic phase, containing dichloromethane and aroma molecules, from the aqueous phase. The organic phase was first extracted, and an additional 500 µL of dichloromethane was added to the aqueous phase for a second extraction. After vortexing and centrifugation under the same conditions, the second organic phase was collected and combined with the first extract. To remove any residual water, a few milligrams of sodium sulfate were added to the organic phase. Finally, 2 µL of the extract was injected into the GC/MS using the same protocol as described previously, with the only modification being a 30-s injection time in splitless mode. Compound identification was

performed following the same procedure as outlined above.

## 2.9. Statistical analyses

ANOVA analyses were conducted to determine significant effects among the examined factors at a significance level of *p* ≤ 0.05. Following this, *post hoc* tests, including Tukey, REGWQ, and bilateral Dunnett, were performed to identify specific differences between conditions.

# 3. Results

## 3.1. Isothiocyanates inhibit ALDH3A1 in a time-dependent manner

Eight isothiocyanates (ITCs) commonly found in food were selected for this study: allyl-isothiocyanate (A-ITC), benzyl-isothiocyanate (B-ITC), butyl-isothiocyanate (BU-ITC), erucin, hexyl-isothiocyanate (HEX-ITC), iberin, 2-phenethyl-isothiocyanate (PE-ITC), and propyl-isothiocyanate (PRO-ITC). These compounds are widely present in various foods and exhibit multiple health benefits (supp. Table 1). The enzymatic activity assay was performed using octanal, a preferred odorant substrate of ALDH3A1, which is metabolized into octanoic acid (Boichot et al., 2023). The presence of ITCs reduced ALDH3A1 activity in a manner dependent on ITC concentration, incubation time, and molecular structure (Fig. 1A and B). Longer incubation times resulted in stronger inhibition, indicating a time-dependent effect. At 100 µM, inhibition levels were markedly higher than at 10 µM for all compounds, confirming a dose-dependent effect. Longer incubation (15 min) further enhanced inhibition, consistent with a progressive covalent modification of the enzyme. Among aliphatic ITCs (PRO-ITC, BU-ITC, and HEX-ITC), inhibitory potency increased with chain length. At 100 µM, all tested ITCs significantly reduced enzymatic activity within 15 min of incubation (Fig. 1B). PRO-ITC and A-ITC, both featuring a three-carbon chain, were the least effective inhibitors. Notably, the double bond in A-ITC did not enhance inhibition compared to PRO-ITC, which lacks this double bond. Among the tested compounds, B-ITC emerged as the most potent inhibitor, inhibiting more than 60% activity of ALDH3A1 at 10 µM and nearly completely inhibiting it after 15 min, and achieving total inhibition after 3 min at 100 µM (supp. Table 2). More broadly, the presence of an aromatic group, as in B-ITC and PE-ITC, increased inhibitory efficacy. Iberin and erucin, both containing a sulfur atom, exhibited inhibitory effects comparable to ITCs with similar chain lengths. Overall, the results indicate that higher ITC concentrations and longer incubation times consistently lead to stronger ALDH3A1 inhibition.

The effect of the most potent inhibitor, B-ITC, was examined against the enzymatic activity of whole saliva. Similar to its impact on recombinant ALDH3A1, B-ITC at 100 µM with 15 min of pre-incubation completely inhibited ALDH activity (Fig. 2A). This finding aligns with the literature, which identifies ALDH3A1 as the primary contributor to salivary ALDH activity (Giebultowicz et al., 2009). To further investigate the inhibition mechanism of B-ITC, kinetic assays were performed without pre-incubation, allowing an assessment of inhibition in the initial moments of the reaction. Michaelis-Menten curves were generated to calculate the *K*<sub>M app</sub>, and Lineweaver-Burk plots were constructed (Fig. 2B). The trend lines intersected at the y-axis, indicating an unchanged maximum reaction rate, while the *K*<sub>M app</sub> increased with B-ITC concentration. These observations confirm a competitive inhibition mechanism of ALDH3A1 by B-ITC, with a *K*<sub>i</sub> of 7.3 ± 4.5 µM. Competitive inhibition suggests that B-ITC binds to the active site, directly competing with octanal at the beginning of the reaction. From our previous work, it is known that the catalytic Cys243, located in the active site, forms a covalent bond with octanal during catalysis (Boichot et al., 2023). Given that prolonged incubation with B-ITC led to stronger inhibition (a trend also observed with other ITCs tested), we hypothesized that a covalent interaction could occur between Cys243 and ITCs.
<caption>**Table 1**</caption>

**Identification of the Cys243 reacting with allyl-isothiocyanate.** Detection of each cysteine modification is indicated by "+", while absence of modification is indicated by "-". The protein was treated with iodoacetamide alone or following a pre-incubation with allyl-isothiocyanate (A-ITC). The numbers in parentheses represent the number of MS/MS spectra identifying the peptide ²³⁶FMNSGQTCVAPDYILCDPSIQNQIVEK²⁶², containing the modified cysteine. Residue numbering corresponds to the natural protein sequence, excluding the initial methionine and the 14-residue N-terminal tag, as annotated in UniProt entry P30838 (https://www.uniprot.org/).

<table><thead><tr><th rowspan="2">Experimental conditions</th><th colspan="4">Type of adduct and Cysteine identification</th></tr><tr><th>Cys243 - Carbamidomethylation</th><th>Cys251 - Carbamidomethylation</th><th>Cys243 - Allyl-isothiocyanate</th><th>Cys251 - Allyl-isothiocyanate</th></tr></thead><tbody><tr><td>Iodoacetamide</td><td>+ (3)</td><td>+ (3)</td><td>- (0)</td><td>- (0)</td></tr><tr><td>Iodoacetamide after A-ITC incubation</td><td>+ (7)</td><td>+ (9)</td><td>+ (2)</td><td>- (0)</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Inhibition of ALDH3A1 enzymatic activity by isothiocyanates. The histograms represent inhibition percentages of ALDH3A1 activity toward octanal. (A) Measurements with 10 µM of isothiocyanate. (B) Measurements with 100 µM of isothiocyanate. The white bars represent the incubation time of 3 min, and the black bars represent the incubation time of 15 min. Standard errors are represented on each column corresponding to the average of the three repetitions. The * symbols indicate a significant difference between the ITC-incubated group and the control condition (without ITC) with a 99% confidence interval. The conducted statistical analyses involved an initial ANOVA followed by a two-sided Dunnett's test.</figcaption></figure>

Initial kinetic data are consistent with a reversible competitive inhibition model, suggesting initial binding of B-ITC to the active site. However, time-dependent inhibition and covalent modification of Cys243 indicate that the final mechanism is an irreversible, covalent inactivation. This suggests a two-step mechanism: initial reversible binding followed by irreversible covalent modification. To further explore the molecular interactions between ALDH3A1 and ITCs, we employed mass spectrometry and X-ray crystallography.

## 3.2. Allyl-isothiocyanate adducts on Cys243 inactivating ALDH3A1 activity

We tried to obtain complex crystal structures of ALDH3A1 with B-

ITC and A-ITC using both co-crystallization and soaking strategies. Among the different approaches, we successfully obtained a complex with A-ITC using a soaking strategy (Fig. 3). Crystals of the complex were found to be isomorphous to the apo ALDH3A1 crystals, and no major conformational changes were observed between the corresponding structures. The catalytic Cys243 is positioned at the core of both subunits of the dimer, and electron density maps clearly indicate that A-ITC is covalently bound to the Cys243 side chains of both subunits A and B (Fig. 3A). This observation confirms a direct interaction between A-ITC and Cys243 through covalent bonding. Notably, no additional electron density was detected around other cysteine residues, suggesting that Cys243 is the primary reactive site for A-ITC binding. The resulting dithiocarbamate moiety (supp. Fig. 1) is stabilized within the active site
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** B-ITC inhibitory effect on aldehyde dehydrogenase activity of human saliva (A) and of recombinant ALDH3A1 (B). (A) Absorbance signal of 3 saliva samples mixed with 100 µM of octanal and 1 mM NAD were measured at 340 nm after 60 min of incubation at 37 °C with (horizontally striped columns) or without (black columns) the addition of 100 µM of B-ITC. White columns correspond to heat-inactivated saliva (90 °C, 10 min) as control. Each measurement was performed in triplicate. Statistical analysis of the results was conducted using ANOVA followed by a Tukey's (HSD) test at a 99% confidence level; two different letters indicate a significantly different result between two conditions. (B) Lineweaver-Burk plot showing double inverse representation of initial rates of the reaction as a function of octanal concentration. Three concentrations of B-ITC were used, 10, 50, and 100 µM. Standard errors were calculated and are represented on each point corresponding to the average of the three repetitions.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Crystal structure of ALDH3A1 with Cys243 covalently bonded with allyl-isothiocyanate. The upper panel (A) shows the ALDH3A1 homodimer colored by chain in cyan (subunit A) and magenta (subunit B) and with the allyl-isothiocyanate ligands bound to Cys243 side chains and represented as sticks and spheres. On the lower panel (B), an enhanced view of the allyl-dithiocarbamate group bound on Cys243 from subunit A (orange sticks and spheres) is shown with surrounding side chains shown as green sticks. 2mFo-DFc electron density map calculated with Phenix and contoured at 1.0 σ is shown around the covalent inhibitor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

through hydrophobic interactions with the side chains of residues Asn114, Tyr115, Thr242, Ile394, and Phe401 (Fig. 3B). Aldehyde substrates also form a covalent intermediate with Cys243 during their conversion into carboxylic acids, this suggests that ITCs compete with aldehydes for binding to the catalytic center. To further investigate the binding mode, we compared our structure with the previously solved

ALDH3A1 complex with the Aldi-1 covalent inhibitor (PDB code: 3SZB). This inhibitor (3-(1-azepanyl)-1-phenyl-1-propanone hydrochloride) was previously found to undergo a beta-elimination generating a vinyl ketone intermediate that covalently modifies the Cys243 (Khanna et al., 2011). Structural superimposition revealed a similar binding pattern of both inhibitors (supp. Fig. 2), reinforcing the idea that ITCs interact with ALDH3A1 in a manner analogous to known covalent inhibitors after an incubation time. To obtain a putative structural model of the ALDH3A1: B-ITC complex, we used our crystallographically determined structure of ALDH3A1 covalently bound to A-ITC as a template (supp. Fig. 3). The common thiocarbamate moiety of B-ITC was kept in the experimentally validated orientation, maintaining the same covalent linkage to the catalytic cysteine 243. The resulting binding mode showed no major steric clashes and remained compatible with the geometry of the active site. Compared with A-ITC, the modeled B-ITC moiety is stabilized by the same surrounding residues as in the A-ITC complex and forms additional hydrophobic contacts with the side chains of Glu61, Met237, Val244 and I391. These additional interactions provide a structural rationale for the stronger inhibitory potency of B-ITC relative to A-ITC observed in our enzymatic assays.

Mass spectrometry experiments were carried out on ALDH3A1 incubated with A-ITC to confirm the results in solution in comparison of the results presented for a protein crystal. Mass spectrometry experiments showed that tryptic digestion produced a peptide 236–262 containing Cys243 and Cys251. As shown in Table 1, both Cys243 and Cys251 were carbamidomethylated following treatment with iodoacetamide alone, as indicated by the detection of three MS/MS spectra for each modified peptide. This confirms that both cysteine residues are accessible and possess free thiol groups under native conditions. However, when the protein was pre-incubated with A-ITC prior to iodoacetamide treatment, differential reactivity was observed. Cys243 exhibited partial modification by A-ITC, evidenced by the detection of two spectra corresponding to the ITC adduct (presumably following a nucleophile addition forming a dithiocarbamate as presented in supp. Fig. 4), while seven spectra still identified the carbamidomethylated form. In contrast, Cys251 showed no evidence of A-ITC modification, with nine spectra corresponding exclusively to the carbamidomethylated peptide and none to the ITC adduct. These results suggest that Cys243 is reactive
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Analysis of the octanal metabolization in the liquid phase. (A) Octanoic acid production in the liquid phase. (B) Octanal quantity resting in the liquid phase after metabolization. The labels S1, S2, S3 indicate the three tested saliva and ALDH3A1 indicates the experiment with the recombinant ALDH3A1. The black columns are the negative control without saliva or enzyme; the horizontally striped columns are the conditions containing saliva (500 µL) or recombinant ALDH3A1 (250 nM), NAD (1 mM), and octanal (500 µM); the dotted columns are the condition supplemented with A-ITC (100 µM); and the vertically striped columns are the condition supplemented with B-ITC (100 µM). Each condition was realized 4 times. Averages and standard errors were calculated and represented on each column. ANOVA followed by a Tuckey (HSD) at a 95% confidence level were realized using XLSTAT. Different letters indicate a significative difference between two conditions.</figcaption></figure>

toward A-ITC, likely due to a more favorable chemical environment and/or higher accessibility, whereas Cys251 does not interact with A-ITC under the same conditions. This highlights a site-specific reactivity of the catalytic Cys243 residue toward A-ITC, consistent with our crystallographic data.

### 3.3. Isothiocyanates inhibit salivary ALDH activity thus modifying the aroma compounds released

It is well established that *in vivo* enzymatic activity within the oral and nasal cavities can rapidly metabolize odorous compounds, generating new metabolites within seconds. These metabolites can exceed the perception threshold, leading to modulation of olfactory receptors activation (Ijichi et al., 2019; Robert-Hazotte et al., 2022). ALDH3A1 has been shown to metabolize odorous molecules, particularly aldehydes, which are proposed to be converted into their corresponding carboxylic acids (Boichot et al., 2023). However, this metabolic transformation remains hypothetical. In this section, we investigate the metabolite production of ALDH3A1 when acting on octanal and assess the influence of ITCs using a GC approach. To approximate physiological conditions, saliva metabolism assays in the presence of ITCs were performed to explore their effect on flavor release. The results are presented in the following lines. First, we examine the impact of ITCs on octanoic acid production using recombinant ALDH3A1 and saliva samples from

three different donors, illustrating the consequences of ITC exposure on metabolite formation. Next, we analyze the formation of octanol as an alternative metabolite, suggesting the involvement of additional salivary enzymes in octanal metabolism. Finally, we explore the effect of the NAD(H) cofactor redox state on these metabolic pathways.

#### 3.3.1. Isothiocyanates inhibit octanoic acid formation

In the absence of saliva or enzyme, no significant formation of octanoic acid was detected (Fig. 4A), and the initial amount of octanal remained present (Fig. 4B). All three saliva samples and recombinant ALDH3A1 promoted a significant production of octanoic acid, with concomitant depletion of octanal, validating enzymatic oxidation in the saliva and by the recombinant ALDH3A1. Among the saliva samples, S1 and S2 showed higher octanoic acid production compared to S3, also concomitant with an enhanced consumption of octanal compared to S3, suggesting interindividual variability in salivary ALDH activity. Addition of A-ITC led to a moderate but statistically significant reduction of octanoic acid levels in S1 and S2 (*p* < 0.05), whereas this effect was not significant for S3 and for the ALDH3A1 recombinant enzyme. This reduced production of octanoic acid was concomitant with higher level of remaining octanal for S1 only. The presence of B-ITC resulted in an inhibition of octanoic acid production from saliva samples S1 and S2 and with the recombinant ALDH3A1. Once again, this reduced production of octanoic acid by B-ITC was concomitant with a significantly higher level
of remaining octanal for S1 and for the recombinant ALDH3A1. Together, these results demonstrate that (i) octanal oxidation in the oral environment is effectively mediated by salivary ALDH activity, and (ii) ITCs, especially B-ITC, act as efficient inhibitors of salivary ALDH activity, probably due to the inhibition of salivary ALDH3A1. Additionally, the decrease in remaining octanal for S2 and S3 in the presence of saliva and B-ITC, despite the strong inhibition of octanoic acid formation, may indicate the consumption of octanal by salivary enzymes other than ALDH3A1.

### 3.3.2. Other enzyme families are involved in odorant salivary metabolism

Following the addition of octanal, the formation of another potential metabolite, octanol, was monitored both in the liquid (Fig. 5A) and in the headspace phase (Fig. 5B). All three saliva samples showed octanol in the liquid phase and two out of the three saliva samples (S1 and S2) exhibited detectable levels of octanol in the headspace, indicating the presence of a salivary enzymatic activity distinct from ALDH3A1 that is capable of reducing octanal to octanol. The absence of octanol in the headspace for saliva S3 may be explained by its lower octanol concentration in the liquid phase compared to S1 and S2, possibly insufficient to reach detectable levels in the gas phase upon equilibration. The presence of either A-ITC or B-ITC reduced octanol levels in S1 only, both in the liquid phase and in the headspace, whereas no significant effect on octanol production was observed for S2 and S3 in either phase. These results support the existence of an alternative salivary enzymatic activity responsible for the reduction of octanal to octanol, as previously reported (Munoz-Gonzalez et al., 2021). It also indicates that

<figure><img src="image_6.png" /></figure>

**Fig. 5.** Analysis of octanol production. Octanol was measured in the liquid (A) and in the headspace phase (B) of the vials. The labels S1, S2, S3 indicate the three tested saliva. Average and standard errors were calculated and represented on each column. ANOVA followed by a Tuckey (HSD) at a 95% confidence level were realized using XLSTAT. Two different letters indicate significant differences between two conditions. The black columns are the negative control without saliva; the horizontally striped columns are the conditions containing saliva (500 µL), NAD (1 mM), and octanal (500 µM); the dotted columns are the condition supplemented with A-ITC (100 µM); and the vertically striped columns are the condition supplemented with B-ITC (100 µM). Each condition was realized in triplicate. Averages and standard errors were calculated and represented on each column. ANOVA followed by a Tuckey (HSD) at a 95% confidence level were realized using XLSTAT. Two different letters indicate a significant difference between two conditions.

isothiocyanates may modulate this reductase activity as well.

Additional experiments presented in the supplementary data highlight the critical role of the cofactor's redox state in metabolite production (supp. Fig. 5). Both octanol and octanoic acid levels were monitored in the liquid phase after addition of saliva to octanal together with either reduced NADH or oxidized NAD. Addition of saliva alone, without any cofactor, did not increase octanoic acid levels compared to the control but did result in octanol production, suggesting that basal levels of NADH present in saliva are sufficient to drive this enzymatic reaction. As expected, the addition of NADH significantly increased octanol production without affecting octanoic acid levels. Interestingly, the addition of NAD resulted in a significant increase in both octanol and octanoic acid levels. While the increase in octanoic acid is consistent with NAD acting as a cofactor for ALDH3A1, the concomitant rise in octanol was unexpected. This may be explained by the presence of salivary enzymatic systems capable of reducing NAD to NADH, thereby increasing the pool of reduced cofactor available for enzymes such as aldoketoreductases to convert octanal to octanol (Boichot et al., 2023).

## 4. Discussion

During food oral processing, reactive compounds from foods are released into the oral cavity and can interact with salivary proteins and enzymes (Carreira et al., 2020). In this study, using a combination of enzymology, structural biology, and mass spectrometry approaches, we demonstrated that dietary isothiocyanates (ITCs) can interact with and inhibit the salivary aldehyde dehydrogenase ALDH3A1, the main ALDH found in saliva (Giebultowicz et al., 2009). Our *in vitro* experiments with recombinant ALDH3A1 and the best inhibitory compound benzyl-isothiocyanate showed a competitive inhibition when the inhibitor was assayed without preincubation. Noteworthy, most ITCs assayed in this study exhibited an increased inhibition after longer exposure with ALDH3A1. Based on the model compound allyl-isothiocyanate, we showed that after incubation covalent binding occurs on the catalytic Cys243 residue, leading to irreversible inhibition. This covalent adduct corresponds to the formation of a dithiocarbamate group on the cysteine residue, a mechanism previously reported for salivary glutathione transferase alpha 1 interacting with allyl-isothiocyanate (Schwartz et al., 2022). In the present work, the inhibitory potential of ITCs was strongly dependent on the nature of their substituent group, with aromatic and longer-chain aliphatic ITCs exhibiting the highest inhibitory effects. Benzyl-ITC was identified as the most potent inhibitor. Based on the solved structure of ALDH3A1 in complex with allyl-isothiocyanate, the active site of ALDH3A1 provides hydrophobic contacts that appear to favor interactions with ITCs containing hydrophobic substituents. This aligns with previous observations that ALDH3A1 exhibits the highest catalytic efficiency with longer-chain aldehydes (Boichot et al., 2023), confirming that ALDH3A1 has an elongated and hydrophobic active site adapted for such substituent. Given the known reactivity of ITCs with cysteine residues, our study provides the first direct evidence that such adducts can form rapidly at physiological concentrations typically found in food or even lower (e.g., allyl-isothiocyanate in broccoli, watercress, and red cabbage at approximately 80 µg per g of dry weight, corresponding to 800 µM (Fusari et al., 2020)).

ALDH3A1 has been previously detected in the nasal cavity, where it was shown to metabolize various odorant aldehydes into their corresponding carboxylic acids (Boichot et al., 2023). Carboxylic acids are less volatile and have different sensory properties compared to their aldehyde precursors. For example, octanal is characterized by waxy, citrus, and orange notes with a green nuance, whereas its metabolite, octanoic acid, is described as having fatty, waxy, and rancid aroma qualities. Here, our findings confirm that a similar metabolic pathway occurs in saliva. Using GC-MS analysis with human saliva, we demonstrated that ITCs can inhibit the metabolism of aldehydes, modulating the quantity of aroma compounds released in both liquid and gas phases. Previous studies have established that the persistence of aroma
compounds is inversely related to their metabolic transformation by salivary or oral mucosa enzymes (Munoz-Gonzalez et al., 2021). Thus, dietary inhibitors such as ITCs could potentially modulate aroma persistence by limiting aldehyde metabolism. Beyond ALDH3A1, other salivary enzymes, such as glutathione transferases (GSTs), have been reported to interact with ITCs. GSTs can either bind ITCs without catalyzing a reaction or actively metabolize them to their glutathione conjugate (Kolm et al., 1995; Schwartz et al., 2022). Therefore, the in situ inhibitory effects of ITCs on ALDH enzymes likely depend on the presence and activity of other enzymatic systems capable of scavenging or metabolizing ITCs. Moreover, additional enzyme families present in saliva are likely involved in octanal metabolism, some of which may not be affected by ITC inhibition. Recent studies have linked salivary enzymes levels to sensory perception. In ageusic/dysgeusic individuals, a significant decrease in GSTA1 expression in saliva was correlated with taste impairment, suggesting a possible link between enzymatic activity and taste perception (Schwartz et al., 2022). Interestingly, another ALDH isoform, ALDH2, shows a polymorphism associated with sweet taste preference (Kawafune et al., 2020). This suggests that multiple enzymatic systems could play a role in cross-modal interactions between aroma and taste perception. Further research is needed to precise the impact of these metabolic reactions on the sensory perception of food.

A crucial aspect of our findings is the role of cofactors in aldehyde metabolism. Our results indicate that depending on the redox state of cofactor (oxidized NAD versus reduced NADH), different metabolites are generated: octanoic acid and octanol in the presence of NAD and octanol only in the presence of NADH. Since ALDH3A1 does not produce octanol, this observation suggests the involvement of other oxidoreductases, possibly aldoketoreductases, previously identified in the oronasal fluids and tissues (Boichot et al., 2023; Schwartz et al., 2021). Importantly, these enzymes could also be affected by ITC supplementation, as octanol generation was significantly altered for one subject's saliva in our study. Given that food introduces a wide variety of chemical compounds, including cofactors such as NAD(H), metabolic reactions likely occur immediately upon food ingestion and chemical release in saliva. The salivary redox is an important oral physiology parameter reflecting the combined action of several antioxidant compounds, mechanisms and enzymes affecting flavor release and perception (Piombino et al., 2014; Schwartz, Neiers, et al., 2020). Additionally, interindividual variability in salivary enzyme activity is an important factor to consider. ALDH activity in saliva is influenced by various factors such as diet or age, contributing to the metabolic differences between individuals observed in GC-MS analyses. Even within the same individual, ALDH activity fluctuates throughout the day, with approximately 60% of salivary ALDH reported as inactive, presumably due to air oxidation. N-acetylcysteine has been suggested as a potential protective reducing compound against ALDH inactivation, possibly through radical-scavenging activity in the oral cavity or salivary glands (Giebultowicz et al., 2010). If confirmed, such mechanisms could result in higher metabolite concentrations and altered sensory perception. Future studies conducted under real food consumption conditions are needed to clarify the impact of these enzymatic reactions on sensory perception. Further investigation is also required to determine the extent to which dietary compounds, including enzymatic inhibitors but also reducing/antioxidant compounds, can modulate oral enzymatic activity and their implications for flavor perception and food preference.

## 5. Conclusion

This study demonstrates that dietary ITCs irreversibly inhibit the salivary enzyme ALDH3A1 through covalent modification of its catalytic cysteine, thereby altering aldehyde metabolism in saliva and potentially influencing aroma perception. Given that ALDH3A1 activity varies among individuals, factors such as diet or age may modulate the extent of ITC-induced inhibition. Future work should quantify these effects under realistic food consumption conditions and determine how ITC

exposure shapes oral aldehyde turnover and sensory outcomes.

## CRediT authorship contribution statement

**Valentin Boichot:** Writing – original draft, Methodology, Investigation. **Jeanne Chaloyard:** Methodology, Investigation. **Karine Gourrat:** Methodology, Investigation. **Jade Moreno:** Methodology, Investigation. **Jean-Michel Saliou:** Methodology, Investigation, Formal analysis. **Jean-Marie Heydel:** Writing – review & editing. **Francis Canon:** Writing – review & editing, Validation. **Fabrice Neiers:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Data curation, Conceptualization. **Mathieu Schwartz:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors acknowledge beamtime access and staff of the ESRF beamline FIP2-BM07 for assistance with X-ray data collection. MS acknowledges the Agence Nationale de la Recherche (ANR, grant number ANR-22-CE21-0001).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2026.148324.

## Data availability

Data will be made available on request.

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