# The dithiocarbamate pesticides maneb and mancozeb disturb the metabolism of lipids and xenobiotics in an in vitro model of metabolic dysfunction-associated steatotic liver disease

Kilian Petitjean, Giovanna Dicara, Sébastien Bristeau, Hugo Coppens-Exandier, Laurence Amalric, Nicole Baran, Camille C Savary, Anne

Corlu, Pascal Loyer, Bernard Fromenty

► To cite this version:

Kilian Petitjean, Giovanna Dicara, Sébastien Bristeau, Hugo Coppens-Exandier, Laurence Amalric, et al.. The dithiocarbamate pesticides maneb and mancozeb disturb the metabolism of lipids and xenobiotics in an in vitro model of metabolic dysfunction-associated steatotic liver disease. Environmental Toxicology and Pharmacology, 2025, 118, pp.104773. 10.1016/j.etap.2025.104773. hal-05198929

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# The dithiocarbamate pesticides maneb and mancozeb disturb the metabolism of lipids and xenobiotics in an in vitro model of metabolic dysfunction-associated steatotic liver disease

Kilian Petitjean<sup>a,1</sup>, Giovanna Dicara<sup>a</sup>, Sébastien Bristeau<sup>b</sup>, Hugo Coppens-Exandier<sup>a,2</sup>, Laurence Amalric<sup>b</sup>, Nicole Baran<sup>b</sup>, Camille C. Savary<sup>c</sup>, Anne Corlu<sup>a</sup>, Pascal Loyer<sup>a</sup>, Bernard Fromenty<sup>a,*</sup>

## ARTICLE INFO

Edited by Dr. M.D. Coleman

### Keywords:

- Pesticides
- Dithiocarbamates
- Maneb
- Manganese
- Liver
- Steatosis

## ABSTRACT

Pesticides are increasingly recognized to be hepatotoxic, but less is known about their toxicity in metabolic dysfunction-associated steatotic liver disease (MASLD). Herein, differentiated HepaRG cells cultured for 2 weeks without (-FA) or with (+FA) a mixture of fatty acids were treated with different pesticides, including maneb and mancozeb, during the same period. While maneb and mancozeb did not induce neutral lipid accumulation in -FA-HepaRG cells, they worsened steatosis in +FA-HepaRG cells. MnCl₂ treatment reproduced these effects. Maneb or MnCl₂ impaired very low-density lipoprotein (VLDL) secretion and increased fatty acid uptake. Zinc supplementation restored VLDL secretion, reduced fatty acid uptake, and prevented steatosis worsening in +FA-HepaRG cells treated with mancozeb or MnCl₂. Maneb, or MnCl₂, also reduced the mRNA expression and activity of several cytochromes P450 in +FA- and -FA-HepaRG cells. This was associated with impaired biotransformation of diazinon. These findings could have major pathophysiological consequences in dithiocarbamate-exposed individuals with MASLD.

## 1. Introduction

Occupational or environmental exposure to different pesticides is suspected of increasing the risk of developing several types of metabolic disorders, including overweight and obesity (Araújo et al., 2022; Zuo et al., 2022), metabolic syndrome (Lamat et al., 2022), type 2 diabetes (Evangelou et al., 2016; Rebouillat et al., 2022), and hepatic steatosis (Sang et al., 2022; Wahlang et al., 2020), also called fatty liver. It is noteworthy that pesticide exposure can induce liver lesions other than steatosis, such as hepatic cytolysis, cholestasis, and vascular disorders (EASL, 2019; Vilas-Boas et al., 2019).

Steatosis is very common in obese individuals (Brunt et al., 2021) and alcoholic patients (Arab et al., 2023). It can also occur in a

significant number of cases of drug-induced liver injury (Borgne-Sanchez and Fromenty, 2025). A recent nomenclature has introduced the term “steatotic liver disease” (SLD), which encompasses all types of fatty liver disease, regardless of their cause (Rinella et al., 2023). In this nomenclature, steatotic liver disease in obese individuals is now called “metabolic dysfunction-associated steatotic liver disease” (MASLD) instead of “nonalcoholic fatty liver disease” (NAFLD) (Rinella et al., 2023).

In obese individuals, MASLD can be worsened by alcohol and certain xenobiotics, including drugs and environmental contaminants (Fromenty and Roden, 2023; Klaunig et al., 2018; Massart et al., 2022). Therefore, many endogenous and exogenous factors may interact and promote the onset or worsening of steatotic liver disease (Klaunig et al.,

<footnote><sup>a</sup> INSERM, Univ Rennes, INRAE, Institut NUMECAN (Nutrition Metabolisms and Cancer) UMR_A 1341, UMR_S 1317, Rennes F-35000, France</footnote>

<footnote><sup>b</sup> BRGM, 3 avenue Claude-Guillemin - BP 36009, Orléans 45060, France</footnote>

<footnote><sup>c</sup> Univ Angers, CHU Angers, INSERM, CNRS, MINT, SFR ICAT, Angers F-49000, France</footnote>

<footnote>* Correspondence to: INSERM UMR_S 1317, Institut NUMECAN (Nutrition Metabolisms and Cancer), Site Pontchaillou-Villejean, 2 avenue du Professeur Léon Bernard, Rennes 35033, France</footnote>

<footnote>E-mail address: bernard.fromenty@inserm.fr (B. Fromenty).</footnote>

<footnote n="1">¹ Present address: Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA</footnote>

<footnote n="2">² Present address: Biopredic International, Parc d'Activité de la Bretèche Bâtiment A4, 35760 Saint Grégoire, France</footnote>
2018; Larrain and Rinella, 2012; Massart et al., 2022). These multiple interactions are of concern because many overweight or obese individuals are treated with different medications and/or exposed to environmental pollutants. Even more worrisome may be the progression of steatosis to advanced chronic liver disease in a significant number of exposed individuals (Tovoli et al., 2023). Indeed, although steatosis is a benign condition in the short term, it can progress to steatohepatitis, advanced fibrosis, cirrhosis, and hepatocellular carcinoma in the long term (Arab et al., 2023; Borgne-Sanchez and Fromenty, 2025; Brunt et al., 2021).

There is now ample evidence that obesity and MASLD are associated with altered hepatic activity of different xenobiotic-metabolizing enzymes (XMEs), such as cytochromes P450 (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs) (Brill et al., 2012; Cobbina and Akhlaghi, 2017; Smit et al., 2018). In particular, increased CYP2E1 activity and reduced CYP3A4 activity have been frequently reported in obesity and MASLD (Brill et al., 2012; Bucher et al., 2018a; Cobbina and Akhlaghi, 2017; Smit et al., 2018). The mechanisms of XME alterations in these metabolic diseases remain unclear and appear complex. For instance, increased CYP2E1 activity might be related to hyperleptinemia, hyperglucagonemia, insulin resistance, and the consumption of certain saturated fatty acids (Aubert et al., 2011; Begriche et al., 2023; Massart et al., 2021). Nevertheless, there is no information on the downstream signaling pathways involved in CYP2E1 induction during obesity and MASLD. Interestingly, MASLD seems to be associated with impaired hepatic activity of several nuclear receptors involved in xenobiotic metabolism such as the aryl hydrocarbon receptor (AhR), the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) (Cave et al., 2016; Klaunig et al., 2018; Yang et al., 2020). Notably, these nuclear receptors are also involved in lipid and carbohydrate metabolism and may have a role in MASLD pathogenesis (Cave et al., 2016; Klaunig et al., 2018; Yang et al., 2020). These data further demonstrate a close relationship between xenobiotic biotransformation and intermediary metabolism in the liver.

In a recent study conducted in metabolically competent differentiated HepaRG cells and other hepatic cells, we investigated the acute cytotoxic effects of 7 pesticides, namely chlorpyrifos-ethyl (hereafter referred to as chlorpyrifos), dimethoate, diazinon, iprodione, imazalil, maneb, and mancozeb (Petitjean et al., 2024). These 7 pesticides were selected for the following reasons: (1) they are often detected in vegetables and fruits in different areas of the world, including Western Europe, South America, Africa, and India (Caldas et al., 2004; EFSA, 2017; Sharma et al., 2023; Ssemugabo et al., 2022); (2) they belong to different chemical and functional classes (Rizzati et al., 2016); (3) their mixture has been previously reported to induce harmful effects in human mesenchymal stem/stromal cells (Hochane et al., 2017; Foucault et al., 2021). Notably, our data in HepaRG cells showed that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the acute cytotoxicity induced by the mixture of these pesticides (Petitjean et al., 2024). Moreover, our investigations revealed that maneb and mancozeb-induced reactive oxygen species (ROS) overproduction and apoptosis could be attributed to the release of Mn, leading to intracellular Mn overload and zinc (Zn) depletion. Finally, maneb and mancozeb-induced cytotoxicity was prevented by Zn supplementation, thus showing that Mn-containing fungicides are acutely toxic by disrupting Mn and Zn homeostasis (Petitjean et al., 2024).

This study aimed to investigate whether repeated exposure to this mixture of 7 pesticides could alter the metabolism of lipids and xenobiotics in differentiated HepaRG cells. As certain xenobiotics can aggravate MASLD (Fromenty and Roden, 2023; Klaunig et al., 2018; Massart et al., 2022), the effects of these pesticides were studied in HepaRG cells cultured or not with a mixture of stearic and oleic acids. Importantly, we have previously shown that culturing HepaRG cells with these fatty acids provides a valuable *in vitro* model of MASLD, reproducing different metabolic features of steatotic liver disease in

patients, including increased CYP2E1 activity and reduced CYP3A4 activity (Bucher et al., 2018a; Le Guillou et al., 2018). Moreover, a comparison by gene set enrichment analysis (GSEA) between the transcriptome GSE102536 dataset obtained in stearate/oleate-supplemented HepaRG cells (Bucher et al., 2018b) and the GSE61260 dataset obtained from biopsies of obese patients with fatty liver (Horvath et al., 2014) revealed a highly significant correlation (p < 0.001) concerning the upregulated genes (Fernandez-Checa et al., 2021). Lastly, we successfully used this model to study, in the context of MASLD, xenobiotic-induced hepatotoxicity (Bronsard et al., 2024; Bucher et al., 2018a; Le Guillou et al., 2018), hydroxychloroquine biotransformation (Ferron et al., 2021), and SARS-CoV-2 receptor ACE2 expression (Cano et al., 2023).

## 2. Materials and methods

### 2.1. Chemicals and reagents

Chlorpyrifos, dimethoate, diazinon, imazalil, iprodione, maneb, mancozeb, dimethyl sulfoxide (DMSO), oleic acid, stearic acid, palmitic acid, L-carnitine, fatty acid-free bovine serum albumin (BSA), manganese chloride (MnCl₂) and zinc chloride (ZnCl₂) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Chemical Abstracts Service (CAS) numbers of the 7 pesticides are provided in Table 1. William's E medium, Dulbecco's phosphate-buffered saline (PBS) Gibco™, glutamine, penicillin, streptomycin, formaldehyde, Nile red, and Hoechst 33342 dyes were obtained from Thermo Fischer Scientific (Waltham, MA). Radiolabeled [¹⁴C(U)]palmitic acid (31.45 GBq/mmol, 3.7 MBq/mL) and [2-¹⁴C] acetic acid (2.183 GBq/mmol, 37 MBq/mL) were purchased from PerkinElmer (Waltham, MA). For the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analyses, analytical internal standards of diazinon (deuterated diazinon D₁₀, 99.7 %), 2-isopropyl-6-methyl-4-pyrimidinol (IMP ¹³C₄, 99.9 %), chlorpyrifos (deuterated chlorpyrifos D₁₀, 99.9 %) and 3,5,6-trichloro-2-pyridinol (TCP ¹³C₃, 99.3 %) were purchased from Techlab (Saint-Julien-lès-Metz, France). Hydrocortisone hemisuccinate was from Pharmacia & Upjohn (Guyancourt, France). DMSO was used as a solvent for the 7 pesticides to prepare a stock solution of 100 mM, except for diazinon, for which the concentration was 10 mM. In this study, the term "pesticide mixture" referred to the combination of the 7 compounds.

### 2.2. Cell culture and treatments

Native HepaRG cells were cultured as previously described, with minor modifications (Cerec et al., 2007). First, progenitor HepaRG cells were seeded at 2.6 × 10⁴ cells/cm² and were incubated in William's E Medium supplemented with 10 % fetal bovine serum (FBS) (Biosera™ S006R20001, Hyclone™ SH30066.03, v/v:3/1), 1 % glutamine (200 mM), 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, and 50 µM hydrocortisone hemisuccinate. After 2 weeks, cell differentiation was enhanced with 2 % DMSO added to the medium for 2 additional weeks. Information regarding the role of DMSO in the differentiation of liver cells, including HepaRG cells, can be found in previous articles (Dubois-Pot-Schneider et al., 2022; Hofmann et al., 2024; Nikolaou et al., 2016). Two days before treatments, FBS and DMSO concentrations were lowered to 5 % and 1 %, respectively as previously described (Bucher et al., 2018a). Then, differentiated HepaRG cells were exposed every 2 or 3 days for 2 weeks to a medium containing or not stearic and oleic acids (150 µM each) and the pesticides. HepaRG cells cultured or not with stearic and oleic acids will hereafter be referred to as +FA-HepaRG and -FA-HepaRG cells, respectively. All investigations were performed 24 h (h) after the last treatment. Control conditions corresponded to HepaRG cells treated with 1 % DMSO without fatty acids and pesticides. HepaRG cells were always maintained in incubators at 37 °C with 5 % CO₂ and saturating humidity. Investigations were carried out on passages 12–17. The concentrations of pesticides
<table><caption>**Table 1**
CAS number, molecular weight (MW) and ADI of the different pesticides and the mixture.</caption><thead><tr><th>Pesticides</th><th>CAS number</th><th>MW</th><th>ADI (EFSA, 2010)</th><th>ADI - Concentrations and percentages</th><th></th><th></th></tr></thead><tbody><tr><td>Chlorpyrifos</td><td>2921-88-2</td><td>350.6</td><td>mg/kg/day</td><td>mg/L</td><td>µM</td><td>% (µM) of each pesticide in the mixture</td></tr><tr><td>Diazinon</td><td>333-41-5</td><td>304.3</td><td>0.0002</td><td>0.12</td><td>0.342</td><td>4.22</td></tr><tr><td>Dimethoate</td><td>60-51-5</td><td>229.3</td><td>0.001</td><td>0.002</td><td>0.008</td><td>0.10</td></tr><tr><td>Imazalil</td><td>35554-44-0</td><td>297.2</td><td>0.025</td><td>0.012</td><td>0.052</td><td>0.64</td></tr><tr><td>Iprodione</td><td>36734-19-7</td><td>330.2</td><td>0.06</td><td>0.3</td><td>1.009</td><td>12.44</td></tr><tr><td>Maneb</td><td>12427-38-2</td><td>265.3</td><td>0.05</td><td>0.72</td><td>2.181</td><td>26.9</td></tr><tr><td>Mancozeb</td><td>8018-01-7</td><td>271.3</td><td>0.05</td><td>0.6</td><td>2.262</td><td>27.9</td></tr><tr><td>Mixture</td><td></td><td></td><td></td><td>2.354</td><td>8.105</td><td>100</td></tr></tbody></table>

used to treat the cells were extrapolated from the international Acceptable Daily Intake (ADI) (EFSA, 2010), which represents the threshold of safety in humans for lifetime exposure (Hochane et al., 2017; Petitjean et al., 2024). Briefly, the ADI value (mg/kg.bw/day) for each pesticide was converted to mg/L, considering the total absorption of the ingested amount and then its dilution in 5 liters of blood (Hochane et al., 2017; Petitjean et al., 2024). The concentration for each pesticide was then converted to µM (Table 1).

In a few investigations, two batches of primary human hepatocytes (PHH) were used to confirm some results obtained in HepaRG cells. These batches were obtained from the Centre de Resources Biologiques (CRB) Santé de Rennes (institutional review board approval BB-0033-00056). Information regarding the donors for PHH 1 and PHH 2 is provided in Supplementary Table 1. Briefly, PHH were seeded at a density of $3 \times 10^5$ cells/cm$^2$ in the same medium as progenitor HepaRG cells. When cells were adherent, the medium was replaced by the same medium as differentiated HepaRG cells. One day before treatments, FBS and DMSO concentrations were lowered to 5 % and 1 %, respectively. PHH were exposed to fatty acids and treated with manebe, mancozeb, or MnCl$_2$ using the same protocol as for HepaRG cells, except for time exposure, which was shortened to 10 or 7 days for PHH 1 and PHH 2, respectively. Indeed, PHH cultures had to be stopped before significant cell detachment, which was heralded by subtle changes in PHH morphology.

## 2.3. Measurement of cellular ATP levels

Cell viability was assessed by measuring the relative intracellular ATP levels using the CellTiter-Glo Luminescent Cell viability assay (Promega, Charbonnières-les-Bains, France). After pesticide treatment, cells were incubated with the CellTiter-Glo reagent for 10 min. Lysates were transferred to an opaque multi-well plate, and luminescent signals were quantified at 540 nm using a POLARstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Cell viability in treated cells was expressed as the percentage of the luminescent values measured in control cells, which was arbitrarily set as 100 %.

## 2.4. Assessment of neutral lipids and triglyceride quantification

Neutral lipid accumulation was evaluated using the Nile red dye (Greenspan et al., 1985), as previously described (Bucher et al., 2018a), with minor modifications. Briefly, HepaRG cells were washed 3 times with PBS, fixed, and stained with PBS containing 4 % formaldehyde and 10 µg/mL Hoechst 33342 dye for 30 min. After 3 washes with PBS, cells were incubated in PBS containing 0.1 µg/mL Nile red for 30 min. Then, cells were washed 3 times with PBS, and fluorescence intensities were measured using a POLARstar Omega microplate reader (BMG Labtech) with excitation/emission wavelengths at 355/460 nm and 520/590 nm for Hoechst 33342 and Nile red, respectively. Nile red fluorescence intensity values were normalized to Hoechst 33342 fluorescence intensity. Cellular triglyceride levels were quantified using the Triglyceride-Glo™ assay (Promega) and then normalized to the concentration of total proteins assessed by using the Pierce™ BCA Protein Assay Kit (Thermo

Fisher Scientific).

## 2.5. Assessment of fatty acid uptake

Fatty acid uptake was measured by using the Fatty Acid Uptake kit from Sigma-Aldrich. Briefly, HepaRG cells were washed with warm PBS and incubated at 37°C for 1 h in unsupplemented William's E medium. Cells were then washed with warm PBS and incubated with a fluorescent probe derived from dodecanoic acid (TF2-C12) for 1.5 h at 37°C. After 2 washes with PBS, fluorescence intensity was measured using a POLARstar Omega microplate reader (BMG Labtech) with excitation/emission wavelengths at 482/520 nm. TF2-C12 fluorescence intensity values were normalized to total protein content and expressed in comparison to control cells.

## 2.6. Assessment of de novo lipogenesis

De novo lipogenesis (DNL) in HepaRG cells was assessed by measuring newly synthesized radiolabeled lipids from [2-¹⁴C] acetic acid, as previously described (Allard et al., 2021). Briefly, the culture medium was removed, and cells were washed with warm PBS. Next, cells were incubated for 3 h with phenol red-free William's E medium containing 1 % fatty acid-free BSA, 50 µM cold acetic acid, and [2-¹⁴C] acetic acid (1.85 kBq per well, corresponding to 16.9 nM). The medium was then removed, and cells were washed with warm PBS before adding a mixture of hexane/isopropanol (3/2;v/v). Cell culture plates were sealed and incubated for 1 h at room temperature for lipid extraction. After the transfer of the plate content in 200 µL microtubes, hexane and PBS were added to have a hexane/isopropanol/PBS ratio of 6/2/3 (v/v/v). Microtubes were then centrifuged at 1000 g for 5 min, and radiolabeled lipids were counted in the upper phase with a Tri-Carb 4910TR liquid scintillation counter (PerkinElmer). Results were normalized to total protein content and expressed compared to control cells.

## 2.7. Assessment of mitochondrial fatty acid oxidation

Mitochondrial fatty acid oxidation (FAO) was assessed by measuring the acid-soluble radiolabeled metabolites resulting from the mitochondrial oxidation of [¹⁴C(U)]palmitic acid, as recently described (Allard et al., 2021). In brief, the culture medium was removed and cells were washed with warm PBS before the addition of phenol red-free William's E medium containing 1 % fatty acid-free BSA, 100 µM cold palmitic acid, [¹⁴C(U)]palmitic acid (185 Bq per well, corresponding to 117 pM), 1 mM L-carnitine and 1 % DMSO. After 3 h of incubation at 37 °C, perchloric acid (final concentration, 6 %) was added, and plates were centrifuged at 2000 g for 10 min, and the supernatant was counted for [¹⁴C]-labeled acid-soluble β-oxidation products using a Tri-Carb 4910TR liquid scintillation counter (PerkinElmer). These [¹⁴C]-labeled acid-soluble β-oxidation products are deemed to be mainly ketone bodies and, to a lesser extent, intermediates of the tricarboxylic acid cycle (Fromenty et al., 1993). Results were normalized to total protein content and expressed compared to control cells.
## 2.8. Assessment of very low-density lipoprotein secretion

The secretion of very low-density lipoprotein (VLDL) from hepatic cells can be assessed via the measurement of apolipoprotein B 100 (hereafter referred to as apoB) in cell supernatant (Allard et al., 2021; Grünig et al., 2018). Hence, apoB levels were measured in HepaRG cell supernatants using the Human Apolipoprotein B ELISA Pro kit from Abtech (Nacka Strand, Sweden) according to the manufacturer's instructions, as recently described (Allard et al., 2021). Briefly, cell supernatants were centrifuged at 1500 g for 5 min and diluted to 1/4 in Apo ELISA buffer. Samples were then incubated in pre-coated strip plates provided in the kit. After 2 h incubation, samples were removed, and plates were washed and incubated for 1 h with monoclonal anti-human apoB (LDL-11-biotin) antibody. Plates were then washed and incubated with a streptavidin-horseradish peroxidase-conjugated antibody. After the last washing, 3,3',5,5'-tetramethylbenzidine (TMB) was used as a substrate for 15 min, protected from light. The reaction was stopped, and absorbance was measured at 450 nm using a POLARstar Omega microplate reader (BMG Labtech). Finally, apoB levels were calculated utilizing a 4-parameter standard curve and expressed compared to control cells.

## 2.9. Measurement of CYP, UGT and sulfotransferase activities

CYP2E1 activity was measured by determining the generation of chlorzoxazone O-glucuronide from chlorzoxazone with a high-performance liquid chromatography (HPLC) method using an Accucore PFP column (Thermo Scientific, Waltham, MA), as previously reported (Bucher et al., 2018a; Quesnot et al., 2018). Briefly, HepaRG cells were incubated for 6 h at 37°C in phenol red-free and DMSO-free William's E medium containing 300 µM chlorzoxazone. At the end of the incubation, aliquots of culture medium were collected and chlorzoxazone O-glucuronide was measured by HPLC analysis with UV detection at 287 nm. CYP2E1 activity was expressed as pmol/mg protein/h.

CYP3A4/5, CYP2B6, CYP1A2, CYP2C19, UGT and sulfotransferase (SULT) activities were respectively measured by the production of hydroxymidazolam, hydroxybupropion, acetaminophen, 4'-hydroxymephenytoin, acetaminophen glucuronide and acetaminophen sulfate using liquid chromatography (LC) coupled to mass spectrometry (MS), as previously described (Anthérieu et al., 2010), with some modifications. Briefly, HepaRG cells were washed at the end of the 2-week treatments with warm PBS and incubated for 30 min in a red phenol-free William's E medium at 37°C. This step aimed at removing all pesticide metabolites synthesized during the 2-week treatment. Next, the medium was replaced by a red phenol-free William's E containing 100 µM midazolam, 100 µM bupropion, 200 µM phenacetin, 100 µM S-mephenytoin, or 1 mM acetaminophen. After 1 h at 37 °C, supernatants were then collected and diluted to 1/2 in acetonitrile in a 96-well plate and kept at -20 °C until analysis. After 10 min of shaking, samples were directly injected into the Exion LC AC system (SCIEX, Toronto, Canada). Compounds were separated on an Xselect HSS T3 column (2.1 × 75 mm, 2.5 µM, Waters, Milford, MA) maintained at 35 °C. The mobile phase was composed of 2 mM ammonium formate containing 0.1 % formic acid and acetonitrile. The total LC run was 5.7 min with a flow rate of 0.5 mL/min. Samples were kept at 4 °C until injection. Detection was operated on the triple quadrupole mass spectrometer AB SCIEX Triple Quad 5500 + (SCIEX) equipped with an electrospray ion source in positive mode. Data processing was performed using Analyst 1.7.2 software (SCIEX). Activities were expressed as nmol/mg protein/h.

## 2.10. Measurement of diazinon, IMP, chlorpyrifos and TCP concentrations

To determine whether HepaRG cell exposure to pesticides could disturb the biotransformation of diazinon to IMP and that of chlorpyrifos

to TCP, levels of these 4 compounds were measured in the culture medium by ultra-performance LC-MS/MS (UPLC-MS/MS). Hence, at the end of the 2-week pesticide treatments, HepaRG cells were washed with warm PBS and incubated for 30 min in a red phenol-free William's E medium at 37°C. This step aimed at removing all pesticide metabolites synthesized during the 2-week treatment. Next, the medium was replaced by a red phenol-free William's E containing 50 µM of diazinon or 50 µM of chlorpyrifos. After 8 and 24 h of incubation at 37°C, the cell culture medium was collected, and HepaRG cells were washed with an equal volume of ultrapure water, which was also collected. The resulting mixtures were centrifuged at 20,000 g for 20 min at 4 °C, and the supernatants were then transferred and frozen until analysis. All compounds were simultaneously quantified in supernatant by UPLC-MS/MS with a Xevo-TQD® triple quadrupole (Waters, Maryville, TN) in MRM (Multiple Reaction Monitoring) mode and with positive electrospray ionization. Chromatographic separation was done with a Waters Acquity UPLC HSS-T3 column (2.1 mm × 100 mm, particle size 1.8 µm) maintained at 30 °C. The mobile phase was a gradient of water/0.01 % formic acid v/v (A) and acetonitrile/0.01 % formic acid v/v (B) with a flow rate of 0.3 mL/min. The gradient started with 95 % eluent A, maintaining isocratic conditions for 0.5 min. Then, eluent B increased to 100 % in 6 min and was maintained for 0.50 min. Finally, initial conditions were reached again in 0.10 min with a re-equilibrium time of 2.9 min to restore the column. The injection volume was 2 µL. For the mass detector, the parameters were as follows: source temperature 150 °C, desolvation temperature 650 °C, cone gas flow 50 L/h, desolvation gas flow 800 L/h, and positive ion mode (spray voltage 2.5 kV).

The injection of analytical standards (at a concentration of 10 µM in water) allowed us to define the analytical features of the compounds and estimate their limit of quantification. Quantification was based on peak area and was performed using the 4 internal standards, namely deuterated diazinon $D_{10}$, IMP $^{13}C_{4}$, deuterated chlorpyrifos $D_{10}$ and TCP $^{13}C_{3}$. To determine the calibration function, injection of 9 concentrations (in a mixture of water/ William's E medium (50/50 v/v)) was performed, ranging from 0.003 to 3 µM for diazinon and IMP and from 0.03 to 30 µM for chlorpyrifos and TCP. Data was best fitted with a quadratic calibration except for IMP (linear function). The limit of quantification was 0.03 µM for each compound. For each series, a control (0.05 µM for diazinon and its metabolite and 5 µM for chlorpyrifos and its metabolite) was injected every 30 samples. The concentrations of the different compounds were normalized by the total protein content. Of note, despite our efforts to measure chlorpyrifos concentrations in the collected biological samples, the quantification of this pesticide was not possible since its concentration was below the limit of quantification.

## 2.11. mRNA expression studies

RNA purification and reverse transcription were performed using the Nucleospin RNA kit from Macherey-Nagel (Düren, Germany) and the High capacity cDNA reverse transcription kit from Applied Biosystems (Woolston, UK), respectively. Quantitative PCR was carried out with the Sybr Green PCR Master Mix from Applied Biosystems on an ABI PRISM 7900HT instrument. The primer sequences are provided in *Supplementary Table 2*. The expression of TATA-box binding protein (TBP) was chosen as a reference and the $2^{\Delta\Delta C_t}$ calculation method was used to express the relative expression of each selected gene.

## 2.12. Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. GraphPad Prism 8 software (GraphPad Software Inc, San Diego, CA) was used for all statistical analyses. Most investigations were carried out by varying two factors, namely fatty acid exposure and treatments with different compounds. For each data set, the Shapiro-Wilk normality test was performed. When this test was positive, comparisons between multiple groups were
performed using a two-way analysis of variance (ANOVA) followed by a *post hoc* Dunnett's test or Sidak's test, as recommended by the GraphPad software. For a few analyses with only one factor (i.e. pesticide treatment), a one-way ANOVA was used, followed by a *post hoc* Dunnett's test. When the data were not normally distributed, a Kruskal-Wallis test was performed, followed by a *post hoc* Dunn's test. The threshold for statistical significance was set to p < 0.05.

## 3. Results

### 3.1. Pesticide-induced cytotoxicity in HepaRG cells after a 2-week exposure

In our recent investigations, pesticide-induced cytotoxicity was assessed in hepatocyte-like HepaRG cells cultured with 2 % DMSO (Petitjean et al., 2024). In the present study, DMSO concentration was lowered to 1 % to curb this cytotoxicity since the primary objective of our investigations was to study lipid and xenobiotic metabolism in differentiated HepaRG cells in a culture condition of reduced cell death. Indeed, previous studies in HepaRG cells consistently showed that high DMSO concentrations close to 2 % significantly enhance the expression and activity of different XMEs including CYPs (Aninat et al., 2006; Dubois-Pot-Schneider et al., 2022; Klein et al., 2014), which explained the previously reported higher pesticide-induced cytotoxicity in HepaRG cells (Petitjean et al., 2024). Nevertheless, culturing HepaRG cells with 1 % DMSO maintains measurable activity of the different CYPs studied herein (i.e. CYP2E1, CYP3A4/5, CYP2B6, CYP1A2, and CYP2C19), as reported below.

In a preliminary series of experiments in -FA and +FA-HepaRG cells, the cytotoxicity of each pesticide or their combination was assessed after 2 weeks of exposure over a wide range of concentrations. Our results showed that at the highest studied concentration (i.e. 3ADI), the reduction of cellular ATP content was never above 30 % in both -FA and +FA-HepaRG cells (Supplementary Figure 1). This 30 % cytotoxicity was brought about only by maneb and mancozeb while the other pesticides induced less or no cytotoxicity at the tested concentrations (Supplementary Figure 1). Notably, the 7-pesticide mixture that included maneb and mancozeb induced a concentration-dependent loss of ATP in -FA and +FA-HepaRG cells, while a mixture without these dithiocarbamates did not induce significant cytotoxicity (Supplementary Figure 1). In addition, the combination of maneb + mancozeb (dithiocarbamates) induced cytotoxicity similar to the one observed with each dithiocarbamate or the mixture (Supplementary Figure 1). Finally, MnCl₂ cytotoxicity was also evaluated because this metal is released in differentiated HepaRG cells during maneb and mancozeb metabolism (Petitjean et al., 2024). However, no significant loss of ATP content was detected for 6.75 µM MnCl₂ (Supplementary Figure 1), which corresponds to the 3ADI concentration of maneb or mancozeb (Table 1). Interestingly, these cytotoxicity data on repeated pesticide treatments (Supplementary Figure 1) are in line with our previous study showing that among the 7 pesticides, only maneb and mancozeb brought about a significant loss of ATP after acute exposure (Petitjean et al., 2024).

### 3.2. Pesticide-induced accumulation of neutral lipids and triglycerides in differentiated HepaRG cells

Next, we wished to determine the steatogenic potential of the 7 pesticides in HepaRG cells using the Nile red dye (Fig. 1A), which allows for assessing the cellular content of neutral lipids (Allard et al., 2021; Bucher et al., 2018a). For this purpose, HepaRG cells cultured for 2 weeks with or without a mixture of stearic and oleic acids were exposed or not to the pesticides during the same period. Five different concentrations of pesticides were studied, namely 1/15, 1/10, 1/5, 1, and 3ADI.

After 2 weeks, neutral lipids in HepaRG cells cultured with fatty acids

(i.e. +FA-HepaRG cells) were moderately but significantly increased as compared to HepaRG cells not exposed to fatty acids (i.e. -FA-HepaRG cells) (Fig. 1B), as previously described (Bucher et al., 2018a; Le Guillou et al., 2018). When cells were exposed to the pesticide mixture, neutral lipids were not increased in -FA-HepaRG cells but were enhanced in +FA-HepaRG cells for the 1/5, 1, and 3ADI concentrations (Fig. 1B). The observed effect in +FA-HepaRG cells was also reproduced when these cells were exposed to maneb, mancozeb, and maneb + mancozeb (dithiocarbamates) at the 1 and 3ADI concentrations (Fig. 1B). The aggravation of steatosis was also observed when cells were exposed to 2.25 and 6.75 µM MnCl₂ (Fig. 1B), respectively, corresponding to the concentrations of Mn brought with the 1 and 3ADI concentrations of the dithiocarbamates maneb and mancozeb. In contrast, neutral lipids did not significantly increase in +FA-HepaRG cells exposed to a mixture not containing the dithiocarbamates (Fig. 1B) and in +FA-HepaRG cells treated only with diazinon, chlorpyrifos, imazalil, dimethoate, or iprodione (Supplementary Figure 2). Finally, neutral lipids were not further increased in +FA-HepaRG cells exposed to ethylene thiourea (Supplementary Figure 2), the main metabolite of the dithiocarbamates maneb and mancozeb (Petitjean et al., 2024).

In MASLD, most neutral lipids accumulating in hepatocytes are made of triglycerides (Ooi et al., 2021; Parlati et al., 2021). Hence, triglycerides were measured in -FA and +FA-HepaRG cells exposed to the pesticide mixture, maneb, mancozeb, or MnCl₂. In keeping with the Nile red assay, triglycerides were not increased in -FA-HepaRG cells exposed to the pesticide mixture, maneb, mancozeb, or MnCl₂ (Supplementary Figure 3). In contrast, exposure to the pesticide mixture, maneb and mancozeb at the 1 and 3ADI concentrations further increased triglyceride accumulation in +FA-HepaRG cells (Supplementary Figure 3). Exposure of +FA-HepaRG cells to MnCl₂ significantly augmented triglyceride accumulation only for 6.75 µM, corresponding to the 3ADI concentration of maneb or mancozeb (Supplementary Figure 3).

Altogether, these results showed that chlorpyrifos, dimethoate, diazinon, iprodione, imazalil, maneb, and mancozeb alone or in a mixture did not induce steatosis in -FA-HepaRG cells after a 2-week exposure. In contrast, maneb, mancozeb, or the pesticide mixture worsened the accumulation of total lipids and triglycerides in +FA-HepaRG cells at the 1 and 3 ADI concentrations. Finally, the exacerbation of steatosis was reproduced when +FA-HepaRG cells were exposed to 2.25 and 6.75 µM MnCl₂, respectively, corresponding to the 1 and 3 ADI concentrations of the dithiocarbamates maneb and mancozeb.

### 3.3. Effects of maneb, mancozeb, and MnCl₂ in PHH cultured with fatty acids

In a small series of investigations, we determined whether maneb, mancozeb, and MnCl₂ could also aggravate neutral lipid accumulation in PHH cultured in the presence of stearic and oleic acids (+FA-PHH). To this end, +FA-PHH were treated at the ADI concentration for maneb and mancozeb or with 2.25 µM MnCl₂, corresponding to the dithiocarbamates ADI. Of note, these experiments did not include PHH not exposed to stearic and oleic acids because of the low number of PHH that could be obtained. Our results for two different batches of PHH (PHH 1 and 2) confirmed that maneb, mancozeb, and MnCl₂ worsen neutral lipid accumulation in +FA-cells, although the effects of these molecules differed between PHH 1 and 2 (Fig. 2).

### 3.4. Mechanisms of dithiocarbamate-induced aggravation of steatosis

To determine the mechanisms whereby dithiocarbamates and MnCl₂ exacerbated steatosis in +FA-HepaRG cells, we investigated the 4 major metabolic pathways that might be potentially involved, namely fatty acid uptake, mitochondrial FAO, DNL, and VLDL secretion.

Fatty acid uptake was not altered by the mixture, maneb, and MnCl₂ after a 24-h treatment (Fig. 3A). However, it was increased by all treatments in both -FA and +FA-HepaRG cells after 48 h, although this
A Staining of neutral lipids with Nile Red

<figure><img src="image_2.png" /><figcaption>With FAs
(+FA)</figcaption></figure>

Untreated

Mixture 1ADI

Mixture 3ADI

B Quantification of neutral lipids

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

(caption on next page)
**Fig. 1.** Effects of pesticides and manganese chloride on neutral lipid accumulation in differentiated HepaRG cells cultured with or without stearic and oleic acids. HepaRG cells were treated with (+FA) or without (-FA) fatty acids, pesticides, or manganese chloride (MnCl₂) for 2 weeks. Pesticide concentrations ranged from 1/ 15–3 ADI, while those of MnCl₂ ranged from 0.15 to 6.75 µM, corresponding to the respective ADI concentrations of maneb and mancozeb. Neutral lipids and nuclei were stained with the fluorescent dyes Nile red and Hoechst 33342, respectively. (A) Representative pictures of -FA and +FA-HepaRG cells exposed or not to the pesticide mixture at the ADI and 3ADI concentrations. Neutral lipids and nuclei are visualized in green and blue, respectively. Pictures were taken at a magnification of × 20. (B) Quantification of neutral lipids in -FA and +FA-HepaRG cells treated with pesticides or MnCl₂ using the Nile red/Hoechst fluorescence intensity ratio. The mixture contained chlorpyrifos, dimethoate, diazinon, imazalil, iprodione, maneb and mancozeb. HepaRG cells treated with dithiocarbamates were exposed to a mixture containing only maneb and mancozeb. Cells were also treated with a mixture that did not contain maneb and mancozeb (mixture w/o dithiocarbamates). Results are means ± SEM for 5–6 independent cultures and are expressed as percentages of the values obtained in untreated -FA-HepaRG cells. *Significantly different from the corresponding untreated or treated -FA-HepaRG cells. #Significantly different from the corresponding untreated -FA or +FA-HepaRG cells (Ctrl).

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

## A Staining of neutral lipids with Nile Red

<figure><img src="image_7.png" /></figure>

## B Quantification of neutral lipids

<figure><img src="image_8.png" /></figure>

**Fig. 2.** Effects of maneb, mancozeb and manganese chloride on neutral lipid accumulation in two different batches of primary human hepatocytes cultured with stearic and oleic acids. Primary human hepatocytes (PHH) cultured with fatty acids (+FA) were treated or not at the ADI concentration for maneb and mancozeb, or with 2.25 µM manganese chloride (MnCl₂), which corresponds to the ADI of the dithiocarbamates. Neutral lipids and nuclei were stained with the fluorescent dyes Nile red and Hoechst 33342, respectively. (A) Representative pictures of +FA-PHH exposed or not to maneb, mancozeb, or MnCl₂. Neutral lipids and nuclei are visualized in green and blue, respectively. Pictures were taken at a magnification of × 20 or ×10, for PHH 1 and PHH 2, respectively. (B) Quantification of neutral lipids in +FA-PHH treated with maneb, mancozeb, or MnCl₂ using the Nile red/Hoechst fluorescence intensity ratio. Results are means ± SEM for 4–6 technical replicates and expressed as percentages of the values obtained in untreated +FA-PHH. *Significantly different from untreated +FA-PHH (Ctrl).

effect was statistically significant only in +FA-HepaRG cells treated with the mixture or maneb at the ADI concentration (Fig. 3A). We also found that the mixture and maneb profoundly reduced VLDL secretion in both -FA and +FA-HepaRG cells after 24 and 48 h of treatment (Fig. 3B). This effect was reproduced by 2.25 µM MnCl₂, corresponding to the ADI concentration of maneb.

Mitochondrial FAO was significantly reduced in +FA-HepaRG cells treated for 2 weeks by the pesticide mixture but only at the 3 ADI

concentration (Supplementary Figure 4). In addition, this metabolic pathway was not altered in -FA and +FA-HepaRG cells treated with different concentrations of maneb, mancozeb, or MnCl₂ (Supplementary Figure 4). DNL was significantly reduced in +FA-HepaRG cells compared to -FA-HepaRG cells (Supplementary Figure 5), confirming that this metabolic pathway is strongly inhibited when fatty acids or lipids are provided in excess (Allard et al., 2021; Ren et al., 2012). DNL was also reduced in -FA-HepaRG cells treated with the mixture, maneb,
## A Fatty acid uptake

<figure><img src="image_9.png" /></figure>

## B VLDL secretion

<figure><img src="image_10.png" /></figure>

**Fig. 3.** Effects of the pesticide mixture, maneb and manganese chloride on fatty acid uptake and VLDL secretion in differentiated HepaRG cells cultured with or without stearic and oleic acids. HepaRG cells were treated with (+FA) or without (-FA) fatty acids, pesticides, or manganese chloride ($MnCl_2$) for 24 or 48 h. Cells were treated at the ADI concentration for the pesticide mixture and maneb, or with 2.25 µM $MnCl_2$, which corresponds to the ADI of maneb. (A) Fatty acid uptake was measured using the fluorescent probe TF2-C12 after 24 or 48 h of treatment. Results are means ± SEM for 3 independent cultures and are expressed as fluorescence intensity/mg protein. \#Significantly different from the corresponding untreated +FA-HepaRG cells (Ctrl). (B) VLDL secretion was assessed by apoB measurement in the culture medium after 24 or 48 h of treatment. Results are means ± SEM for 3–6 independent cultures and are expressed as percentages of the values obtained in untreated -FA-HepaRG cells. \#Significantly different from the corresponding untreated -FA and +FA-HepaRG cells (Ctrl).

mancozeb, or $MnCl_2$, but these treatments did not alter DNL in +FA-HepaRG cells (Supplementary Figure 5).

Altogether, these results suggested that the pesticide mixture, maneb, and $MnCl_2$ could exacerbate steatosis in +FA-HepaRG cells by rapidly impairing VLDL secretion and enhancing fatty acid uptake. Of note, the increase (although not significant) in fatty acid uptake in -FA-HepaRG cells treated with the mixture, maneb, or $MnCl_2$ (Fig. 3A) appeared to have no impact on neutral lipids in -FA-HepaRG cells (Fig. 1B). This was most probably because these cells were cultured without stearic and oleic acids.

### 3.5. Effect of $ZnCl_2$ supplementation on neutral lipids, VLDL secretion and fatty acid uptake

In our recent study, maneb-, mancozeb-, and $MnCl_2$-induced acute cytotoxicity in differentiated HepaRG cells was significantly prevented by $ZnCl_2$ supplementation (Petitjean et al., 2024). Importantly, $ZnCl_2$

supplementation reduced (although not significantly) the intracellular levels of $MnCl_2$ and significantly restored the depletion of cellular Zn induced by maneb and $MnCl_2$ (Petitjean et al., 2024). Hence, in this study, we wished to determine whether Zn supplementation could also prevent the exacerbation of steatosis in +FA-HepaRG cells induced by a 2-week treatment of mancozeb and $MnCl_2$. Both 22.5 and 45 µM $ZnCl_2$ significantly alleviated the accumulation of neutral lipids in +FA-HepaRG cells treated with mancozeb at the ADI concentration or with 2.25 µM $MnCl_2$, corresponding to the ADI of mancozeb (Fig. 4A). Moreover, 45 µM $ZnCl_2$ partially alleviated the reduction of VLDL secretion induced by maneb, mancozeb, and $MnCl_2$ (Fig. 4B), and fully prevented the increase in fatty acid uptake induced by these molecules in +FA-HepaRG cells after 48 h of treatment (Fig. 4C). Taken together, these results suggested that maneb, mancozeb, and $MnCl_2$ could disturb lipid metabolism and worsen steatosis in +FA-HepaRG cells by disrupting Mn and Zn homeostasis.
A Neutral lipids

<figure><img src="image_11.png" /></figure>

B VLDL secretion

<figure><img src="image_12.png" /></figure>

C Fatty acid uptake

<figure><img src="image_13.png" /></figure>

(caption on next page)
**Fig. 4.** Effects of zinc supplementation on maneb, mancozeb and manganese chloride-induced changes in neutral lipids, VLDL secretion and fatty acid uptake in differentiated HepaRG cells cultured with stearic and oleic acids. HepaRG cells cultured with fatty acids (+FA-HepaRG cells) were treated with or without zinc chloride (ZnCl₂), mancozeb, maneb, or manganese chloride (MnCl₂) for 48 h or 2 weeks. Cells were treated at the ADI concentration for mancozeb and maneb, or with 2.25 µM MnCl₂, which corresponds to the ADI of these dithiocarbamates. Zinc supplementation was achieved with 22.5 or 45 µM ZnCl₂. (A) Neutral lipids. Neutral lipids were assessed after 2 weeks of treatment using the Nile red/Hoechst fluorescence intensity ratio. Results are expressed as percentages of the values obtained in untreated +FA-HepaRG cells. (B) VLDL secretion. VLDL secretion was assessed by apoB measurement in the culture medium after 48 h of treatment. Results are expressed as percentages of the values obtained in untreated +FA-HepaRG cells. (C) Fatty acid uptake. Fatty acid uptake was measured using the fluorescent probe TF2-C12 after 48 h of treatment. Results are expressed as fluorescence intensity/mg protein. Results for (A), (B) and (C) are means ± SEM for 3–4 independent experiments. *Significantly different from the corresponding +FA-HepaRG cells not treated with ZnCl₂. #Significantly different from the corresponding +FA-HepaRG cells unexposed to dithiocarbamates or MnCl₂ (Ctrl).

### 3.6. Expression of genes involved in VLDL secretion and fatty acid uptake

In the next series of investigations, the mRNA expression of different genes involved in VLDL secretion and fatty acid uptake was measured in -FA and +FA-HepaRG cells treated for 24 and 48 h with the pesticide mixture or maneb at the ADI concentration, or with 2.25 µM MnCl₂, corresponding to the ADI of maneb. Regarding genes involved in VLDL secretion, mRNA levels of apolipoprotein B (APOB), apolipoprotein C3 (APOC3), and microsomal triglyceride transfer protein (MTTP) were reduced by the different treatments after 48 h in -FA and +FA-HepaRG cells, although to different extents (Fig. 5A). However, APOB and APOC3 expression were less or not affected after 24 h (Fig. 5A). Because reduced VLDL secretion can be associated with endoplasmic reticulum (ER) stress (Allard et al., 2021; Ota et al., 2008), mRNA levels of DDIT3 (CHOP), ERN1 (IRE1α), EIF2AK3 (PERK), HSPA5 (BIP) and ATF6 were measured in -FA and +FA-HepaRG cells treated for 24 and 48 h with the pesticide mixture or maneb at the ADI concentration, or with 2.25 µM MnCl₂, corresponding to the ADI of maneb. However, the mRNA expression of these different genes was not significantly increased after 24 and 48 h (data not shown), thus suggesting that ER stress was not involved in the impairment of VLDL secretion induced by the pesticide mixture, maneb and MnCl₂ (Fig. 3A).

Regarding genes involved in fatty acid uptake, mRNA levels of CD36 molecule (fatty acid transporter, FAT/CD36), fatty acid transport protein 2 (FATP2) and fatty acid transport protein 4 (FATP4) were either reduced or unchanged by the different treatments after 24 and 48 h in -FA and +FA-HepaRG cells (Fig. 5B). Hence, these results suggested that higher fatty acid uptake in HepaRG cells treated for 48 h with the pesticide mixture, maneb, or MnCl₂ (Fig. 3A) could not be explained by the observed changes in the expression of these key fatty acid transporters in hepatocytes.

### 3.7. Activity and expression of phase I and II XMEs

In the next series of investigations, the activity and expression of several XMEs were measured for two reasons. First, numerous xenobiotics, including pesticides, have been shown to modulate the expression and/or activity of many XMEs (Abass et al., 2012; Amacher, 2010; Hakkola et al., 2020). Second, MASLD has been consistently associated with altered expression and/or activity of different CYPs, UGTs, and SULTs (Brill et al., 2012; Cobbina and Akhlaghi, 2017; Smit et al., 2018), as previously mentioned. Notably, preliminary investigations in -FA and +FA-HepaRG cells showed that the pesticide mixture and maneb significantly reduced XME activity at concentrations much lower than 1ADI with reproducible effects for the 1/10ADI concentration. Hence, this low concentration was kept for the remaining investigations reported below.

Regarding the exposure to fatty acids alone, our data showed that CYP2E1 activity and mRNA expression were increased in +FA-HepaRG cells (Fig. 6A, Supplementary Table 3), whereas CYP3A4 activity and mRNA expression were reduced in these cells (Fig. 6B, Supplementary Table 3), which was in line with previous data in experimental and human MASLD (Aubert et al., 2011; Begriche et al., 2023; Brill et al., 2012; Bucher et al., 2018a; Cobbina and Akhlaghi, 2017; Smit et al., 2018). In contrast, the activity of CYP2B6 (Fig. 6C, Supplementary

Table 3) and that of CYP1A2, CYP2C19, UGT, and SULT (Supplementary Figure 6) were unchanged in +FA-HepaRG cells as compared with -FA-HepaRG cells.

Concerning pesticide and MnCl₂ exposure, a reduction of the activity and mRNA expression of CYP2E1, CYP3A4, and CYP2B6 was found in -FA and +FA-HepaRG cells exposed to the pesticide mixture, maneb or MnCl₂, although to a different extent depending on the experimental conditions (Fig. 6). Notably, the reduction of CYP3A4 and CYP2B6 activity and expression was more pronounced in +FA-HepaRG cells compared to -FA-HepaRG cells (Figs. 6b and 6c). In contrast, the activity and mRNA expression of CYP2E1, CYP3A4, and CYP2B6 were less or not altered with a mixture not containing the dithiocarbamates maneb and mancozeb (Fig. 6). Finally, the pesticide mixture, maneb, and MnCl₂ did not decrease the activity of CYP1A2, UGT, and SULT (Supplementary Figure 6). However, a significant reduction in CYP2C19 activity was observed only in +FA-HepaRG cells incubated with the pesticide mixture, maneb, or the pesticide mixture not containing the dithiocarbamates (Supplementary Figure 6). Altogether, these data showed that the pesticide mixture, maneb and MnCl₂, reduced the expression and activity of CYP2E1, CYP3A4, and CYP2B6, but not that of CYP1A2, UGT, and SULT. The decreased activity of CYP2E1, CYP3A4, and CYP2B6 could be due to a transcriptional or post-transcriptional mechanism.

### 3.8. Expression of genes encoding nuclear receptors

The nuclear receptors AhR, CAR, PXR, and farnesoid X-activated receptor (FXR) play a major role in regulating the expression of many XMEs, including CYPs (Riddick, 2023; Toporova and Balaguer, 2020). In addition, there is growing evidence that these nuclear receptors also play a role in lipid metabolism (Cai et al., 2021; Toporova and Balaguer, 2020; Wang et al., 2022) and MASLD pathophysiology (Cave et al., 2016; Klaunig et al., 2018; Yang et al., 2020). Importantly, HepaRG cells express significant levels of PXR, CAR, FXR, and AhR, which are responsive to different agonizts, antagonists, or other types of modulators (Aninat et al., 2006; Kamaraj et al., 2025; Tanner et al., 2018; Zhang et al., 2020). Hence, we assessed the mRNA expression of AHR, CAR, FXR, and PXR in -FA and +FA-HepaRG cells treated with the pesticide mixture, the mixture without maneb and mancozeb, maneb or MnCl₂ for 2 weeks. Overall, these treatments reduced the mRNA levels of CAR, FXR, and PXR, although to a different extent depending on the experimental conditions (Fig. 7). In contrast, AHR expression was not altered regardless of the experimental conditions (Fig. 7).

### 3.9. Investigations on diazinon and chlorpyrifos metabolism

In a last series of experiments, we wished to determine whether the pesticide mixture, maneb and MnCl₂ could alter the metabolism of the organophosphorothioate insecticides diazinon and chlorpyrifos in -FA and +FA-HepaRG cells since reduced activity of CYP3A4 (Fig. 6B), CYP2B6 (Fig. 6C) and CYP2C19 (Supplementary Figure 6) was found after a 2-week exposure to these treatments. Indeed, diazinon is metabolized to diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol (IMP) by CYP2B6 and CYP3A4 but also by CYP1A2 and CYP2C19 (Kappers et al., 2001; Mutch and Williams, 2006; Sams et al., 2004) (Fig. 8A),
A mRNA expression of genes involved in VLDL secretion

24-hour treatment

<figure><img src="image_14.png" /></figure>

48-hour treatment

<figure><img src="image_15.png" /></figure>

B mRNA expression of genes involved in fatty acid uptake

24-hour treatment

<figure><img src="image_16.png" /></figure>

48-hour treatment

<figure><img src="image_17.png" /></figure>

**Fig. 5.** Effects of the pesticide mixture, maneb and manganese chloride on the mRNA expression of genes involved in VLDL secretion and fatty acid uptake in differentiated HepaRG cells cultured with or without stearic and oleic acids. HepaRG cells were treated with (+FA) or without (-FA) fatty acids, pesticides, or manganese chloride (MnCl₂) for 24 or 48 h. Cells were treated at the ADI concentration for the mixture and maneb, or with 2.25 µM MnCl₂, which corresponds to the ADI of maneb. (A) Expression of *APOB*, *APOC3* and *MTTP* after 24 and 48 h of treatment. (B) Expression of *FAT/CD36*, *FATP2* and *FATP4* after 24 and 48 h of treatment. Results for (A) and (B) are means ± SEM for 3 independent cultures and are expressed as fold change of untreated -FA-HepaRG cells. \*Significantly different from the corresponding treated -FA-HepaRG cells. \#Significantly different from the corresponding untreated -FA or +FA-HepaRG cells (Ctrl).
A CYP2E1 activity and mRNA expression

<figure><img src="image_18.png" /></figure>

B CYP3A4/5 activity and CYP3A4 mRNA expression

<figure><img src="image_19.png" /></figure>

C CYP2B6 activity and mRNA expression

<figure><img src="image_20.png" /></figure>

(caption on next page)
**Fig. 6.** Effects of pesticides and manganese chloride on the mRNA expression and activity of CYP2E1, CYP3A4/5 and CYP2B6 in differentiated HepaRG cells cultured with (+FA) or without (-FA) stearic and oleic acids. HepaRG cells were treated with or without fatty acids, pesticides, or manganese chloride (MnCl₂) for 2 weeks. Cells were treated at the 1/10 ADI concentration for the pesticide mixture (Mix), the mixture without dithiocarbamates (Mix w/o DTC) and maneb, or with 0.22 µM MnCl₂, which corresponds to 1/10 ADI of maneb. (A) CYP2E1 activity and mRNA expression. CYP2E1 activity, assessed by measuring the formation of chlorzoxazone-O-glucuronide, is expressed in pmol/mg protein/h. CYP2E1 expression is expressed as fold change of untreated -FA-HepaRG cells. (B) CYP3A4 activity and mRNA expression. CYP3A4 activity, assessed by the formation of hydroxymidazolam, is expressed in µmol/mg protein/h. CYP3A4 expression is expressed as fold change of untreated -FA-HepaRG cells. (C) CYP2B6 activity and mRNA expression. CYP2B6 activity, assessed by the formation of hydroxybupropion, is expressed in µmol/mg protein/h. CYP2B6 expression is expressed as fold change of untreated -FA-HepaRG cells. Results for (A), (B) and (C) are means ± SEM for 3–7 independent cultures. \*Significantly different from the corresponding untreated or treated -FA-HepaRG cells. \#Significantly different from the corresponding untreated -FA or +FA-HepaRG cells (Ctrl).

<figure><img src="image_21.png" /></figure>

**Fig. 7.** Effects of pesticides and manganese chloride on the mRNA expression of AHR, CAR, FXR and PXR in differentiated HepaRG cells cultured with or without stearic and oleic acids. HepaRG cells were treated with (+FA) or without (-FA) fatty acids, pesticides, or manganese chloride (MnCl₂) for 2 weeks. Cells were treated at the 1/10 ADI concentration for the pesticide mixture (Mix), the mixture without dithiocarbamates (Mix w/o DTC) and maneb, or with 0.22 µM MnCl₂, which corresponds to 1/10 ADI of maneb. Results are means ± SEM for 3 independent cultures and expressed as fold change of untreated -FA-HepaRG cells. \*Significantly different from the corresponding untreated or treated -FA-HepaRG cells. \#Significantly different from the corresponding untreated -FA or +FA-HepaRG cells (Ctrl).

whereas chlorpyrifos is metabolized to 3,5,6-trichloro-2-pyridinol (TCP) and chlorpyrifos oxon also by these different CYPs (Sams et al., 2004; Tang et al., 2001). Hence, diazinon metabolism was assessed in this study after a 2-week exposure of the HepaRG cells to the pesticide mixture, maneb and MnCl₂, by measuring the disappearance of diazinon and the production of IMP after an 8- and a 24-h incubation with 50 µM diazinon. Chlorpyrifos metabolism was studied via the production of

TCP from 50 µM chlorpyrifos after the same incubation times.

Overall, our results showed that the pesticide mixture, maneb and MnCl₂, lessened both the disappearance of diazinon and the production of IMP, and this effect was generally more pronounced in +FA-HepaRG cells as compared to -FA-HepaRG cells (Figs. 8B and 8C). In contrast, these different treatments did not change TCP production from chlorpyrifos regardless of the incubation time (Supplementary Figure 7).
A Schematic metabolism of diazinon

<figure><img src="image_22.png" /></figure>

B Concentrations of diazinon

<figure><img src="image_23.png" /></figure>

C Concentrations of 2-isopropyl-6-methyl-4-pyrimidinol

<figure><img src="image_24.png" /></figure>

**Fig. 8.** Effects of pesticides and manganese chloride on diazinon metabolism in differentiated HepaRG cells cultured with or without stearic and oleic acids. HepaRG cells were treated with (+FA) or without (-FA) fatty acids, pesticides, or manganese chloride (MnCl₂) for 2 weeks. Cells were treated at the 1/10 ADI concentration for the pesticide mixture (Mix) and maneb (Mb), or with 0.22 µM MnCl₂, which corresponds to 1/10 ADI of maneb. (A) Schematic metabolism of diazinon. (B) and (C) Concentrations of diazinon and 2-isopropyl-6-methyl-4-pyrimidinol, respectively. After the 2 weeks, HepaRG cells were treated with 50 µM diazinon and concentrations of remaining diazinon and produced 2-isopropyl-6-methyl-4-pyrimidinol were measured in the culture media after 8 and 24 h. Results for (b) and (c) are means ± SEM for 4 independent cultures and expressed in µM/mg protein. *Significantly different from the corresponding untreated or treated -FA-HepaRG cells. #Significantly different from the corresponding untreated -FA or +FA-HepaRG cells (C).
Hence, these results suggested that decreased activity of CYP3A4 (Fig. 6B), CYP2B6 (Fig. 6C), and CYP2C19 (Supplementary Figure 6) induced by the pesticide mixture, maneb and $MnCl_2$ in -FA and +FA-HepaRG cells was sufficient to alter the biotransformation of diazinon but not that of chlorpyrifos.

## 4. Discussion

This study is the first to show that the dithiocarbamate pesticides maneb and mancozeb can disturb the metabolism of lipids and xenobiotics in differentiated HepaRG cells treated for 2 weeks, possibly via the intracellular release of Mn. Notably, we recently reported that a single treatment of differentiated HepaRG cells with these pesticides induced ROS overproduction and caspase-dependent apoptosis via their biotransformation and the intracellular release of Mn (Petitjean et al., 2024). In the present study, DMSO concentration was 1 % instead of 2 % in our previous investigations (Petitjean et al., 2024). This allowed us to significantly curb dithiocarbamate-induced cytotoxicity in the setting of repeated exposures, possibly by lowering the expression and activity of different XMEs, including CYPs (Aninat et al., 2006; Dubois-Pot-Schneider et al., 2022; Klein et al., 2014). Indeed, the loss of cellular ATP in treated HepaRG cells was never above 30 % of the control cells, even at the highest concentrations of maneb and mancozeb. Altogether, our results indicate that maneb and mancozeb can have broad hepatocellular effects with dire consequences on metabolism, oxidative stress, and cell death. Of note, a maximum of 30 % decrease in ATP content in HepaRG cells did not impede the study of lipid accumulation and related mechanisms (Allard et al., 2021, 2024).

One key finding in this study was the exacerbation of steatosis in +FA-HepaRG cells by maneb, mancozeb, and $MnCl_2$, whereas these molecules did not induce neutral lipid accumulation in -FA-HepaRG cells. Furthermore, our study disclosed that steatosis aggravation was associated with increased fatty acid uptake and reduced VLDL secretion, whereas mitochondrial FAO and DNL were unchanged. Interestingly, impaired VLDL secretion was associated with a reduction in the expression of *APOB* and *APOC3* (two major apolipoproteins in VLDL) and of *MTTP* (also referred to as *MTP*), a key enzyme for the assembly and secretion of this lipoprotein (Allard et al., 2024; Heeren and Scheja, 2021). However, the decrease in VLDL secretion and expression of *APOB*, *APOC3* and *MTTP* were not associated with ER stress, contrary to what occurred with different pharmaceuticals inducing steatosis in HepaRG cells such as 5-fluorouracil, indomethacin, rifampicin, troglitazone (Allard et al., 2021) and busulfan (Allard et al., 2024). Thus, further investigations will be needed to determine the exact mechanism (s) whereby maneb, mancozeb, and $MnCl_2$ reduce *APOB*, *APOC3*, and *MTTP* expression and VLDL secretion in hepatocytes.

A previous study reported that short-term treatment with mancozeb worsened neutral lipid accumulation in HepG2 cells cultured with oleic and linoleic acids (Pirozzi et al., 2016). However, the aggravation of steatosis was observed with concentrations of mancozeb, leading to more than 60 % cell mortality. Thus, it is unclear whether the observed effects on cellular lipids were specific or the mere consequence of severe mitochondrial dysfunction and other major disturbances of cellular homeostasis. In addition, this earlier study did not investigate the effect of mancozeb on fatty acid uptake, mitochondrial FAO, DNL, and VLDL secretion.

In this study, mancozeb and $MnCl_2$-induced worsening of steatosis in +FA-HepaRG cells was fully prevented by $ZnCl_2$ supplementation. In addition, this supplementation restored the impairment of VLDL secretion and alleviated the increase in fatty acid uptake induced by mancozeb, maneb, or $MnCl_2$. Notably, Zn supplementation also reduced ROS overproduction, caspase activation, and cytotoxicity induced by a single exposure to high concentrations of dithiocarbamates (3 ADI) and $MnCl_2$ (7 µM) (Petitjean et al., 2024). Hence, these findings strongly suggest that the dithiocarbamates maneb and mancozeb are toxic to the hepatocytes, at least in part via disruption of Mn and Zn homeostasis, which

can be restored by Zn supplementation. Zn supplementation in individuals exposed to high levels of dithiocarbamates might be an efficient means to prevent their potential hepatotoxicity. To our knowledge, no clinical or epidemiological studies reported dithiocarbamate-induced liver injury. Nevertheless, maneb and mancozeb can cause hepatic damage in rodents in particular via oxidative stress and DNA damage (Aprioku et al., 2023; Gao et al., 2025; Nuchniyom et al., 2023; Saber et al., 2019; Sefi et al., 2019; Zhang et al., 2022a). Interestingly, Zn supplementation was reported to prevent maneb-induced teratogenicity in rats (Larsson et al., 1976) and maneb-induced kidney injury in mice (Sefi et al., 2020), thus suggesting that Zn could prevent dithiocarbamate toxicity in different tissues and organs.

Recent epidemiological studies reported a positive association between blood Mn levels and hepatic steatosis (Liu et al., 2023; Spaur et al., 2022; Tang et al., 2024; Xue and Chen, 2025), although other investigations found that increased dietary Mn intake, or high blood Mn contents might be a potential protective factor for MASLD (Schophaus et al., 2024; Zhang et al., 2022b). Unfortunately, these studies were not designed to determine whether Mn exposure could aggravate preexisting fatty liver. Interestingly, investigations in the yellow catfish showed that Mn excess can induce hepatic steatosis, possibly via increased DNL and decreased mitochondrial FAO (Zhao et al., 2022, 2023). Moreover, Zn deficiency in rats was reported to induce steatosis depending on the dietary fat composition (Eder and Kirchgessner, 1994, 1995; tom Dieck et al., 2005). Mechanistic investigations suggested that hepatic lipid accumulation in Zn-deficient rats could be secondary to increased DNL and reduced mitochondrial FAO (Eder and Kirchgessner, 1995; tom Dieck et al., 2005). However, these experimental studies did not investigate the effects of Mn overload and Zn deficiency in the liver of animals fed a high-fat diet. Of note, our investigations in differentiated HepaRG cells showed that $MnCl_2$ did not induce steatosis in cells cultured without fatty acids (i.e. -FA-HepaRG cells). In addition, $MnCl_2$-induced aggravation of steatosis in +FA-HepaRG cells did not involve DNL and mitochondrial FAO. Taken together, these findings suggest that hepatic steatosis induced by Mn excess and Zn deficiency could be specific to some animal species. Furthermore, the mechanisms whereby Mn overload worsens steatosis in +FA-HepaRG cells might differ from those in which Mn excess leads to hepatic lipid accumulation in non-obese animals.

Previous experimental and clinical studies reported that some xenobiotics can aggravate steatosis in the context of preexisting obesity and MASLD. For instance, this has been observed with different pharmaceuticals, including the antibiotic tetracycline, the antidiabetic rosiglitazone, the hemorrhagic agent pentoxyfylline, and some anti-inflammatory corticosteroids (Allard et al., 2019; Massart et al., 2017, 2022). Environmental toxicants reported to worsen fatty liver in obese rodents include bisphenol A, hexabromocyclododecane, nonylphenol, perchloroethylene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and tetribromodiphenyl ether (Massart et al., 2022; Yanagisawa et al., 2014; Yang et al., 2019). Thus, the present study allows to add maneb, mancozeb, and Mn to the growing number of molecules that could worsen obesity-associated steatosis.

Obesity and MASLD are associated with altered activity of different phase I XMEs, including CYPs (Brill et al., 2012; Cobbina and Akhlaghi, 2017; Leahy et al., 2024; Smit et al., 2018). Accordingly, CYP2E1 activity was increased while CYP3A4/5 activity was decreased in +FA-HepaRG cells in comparison with -FA-HepaRG cells. These results are fully in line with several clinical investigations carried out in obese patients with MASLD (Begriche et al., 2023; Brill et al., 2012; Chalasani et al., 2003; Cobbina and Akhlaghi, 2017; Kolwankar et al., 2007; Smit et al., 2018). In addition to obesity and related metabolic diseases, numerous xenobiotics, including pesticides, can modulate the expression and activity of many XMEs (Abass et al., 2012; Amacher, 2010; Hakkola et al., 2020). In this study, low concentration (1/10 ADI) of maneb significantly reduced the activity of CYP2E1, CYP3A4/5, CYP2B6, and CYP2C19 in -FA and +FA-HepaRG cells. Notably,
pesticides can be metabolized by a large repertoire of XMEs. For instance, diazinon and chlorpyrifos are metabolized by different CYPs, including CYP1A2, CYP2B6, CYP2C19, and CYP3A4 (Kappers et al., 2001; Mutch and Williams, 2006; Sams et al., 2004; Tang et al., 2001).

Hence, there can be numerous interactions between MASLD and pesticide exposure as regards xenobiotic biotransformation. In keeping with this hypothesis, alteration of diazinon biotransformation induced by low concentration of maneb was exacerbated in +FA-HepaRG cells compared to -FA-HepaRG cells. In contrast, chlorpyrifos biotransformation to 3,5,6-trichloro-2-pyridinol was not impaired by maneb. Although we did not have a definite explanation for this difference, chlorpyrifos metabolism might be less dependent on CYP3A4/5, CYP2B6, and CYP2C19 than is diazinon. Importantly, various pharmaceuticals are metabolized by these CYPs (Hedrich et al., 2016; Hirota et al., 2013; Klyushova et al., 2022). Hence, individuals exposed to dithiocarbamates might be at risk for drug-drug interactions and related adverse effects.

CAR and PXR are key nuclear receptors regulating CYP3A4/5 and CYP2B6 expression (Cai et al., 2021; Hedrich et al., 2016; Klyushova et al., 2022). Interestingly, the pesticide mixture, maneb and MnCl₂ decreased CAR and PXR mRNA levels in -FA- and +FA-HepaRG cells, although to a different extent depending on the experimental conditions. Hence, the decrease in CAR and PXR mRNA levels in some experimental settings might explain, at least in part, the reduction of CYP3A4 and CYP2D6 expression. Of note, there is growing evidence that CAR and PXR also regulate the metabolism of carbohydrates and lipids (Cai et al., 2021; Cave et al., 2016). Thus, further investigations would be required to determine whether dithiocarbamate-induced reduction in CAR and PXR expression participates in the aggravation of steatosis in +FA-HepaRG cells. The pesticide mixture and maneb also decreased the expression of *FXR*, which plays a major role in bile acid metabolism (Sun et al., 2021). Interestingly, drug-induced down-regulation of *FXR* signaling favors intrahepatic cholestasis (Ding et al., 2018; Petrov et al., 2021). Hence, individuals exposed to dithiocarbamates might also be at risk for cholestatic liver disease.

Our study has some limitations. First, steatosis was induced in differentiated HepaRG cells using a combination of stearate (C18:0) and oleate (C18:1), as previously reported (Bucher et al., 2018a, 2018b; Le Guillou et al., 2018). While palmitic acid (C16:0) can be used alone or in combination with other fatty acids to induce lipid accumulation in different hepatic cell lines (Ramos et al., 2021; Vergani, 2019), we found it to be more cytotoxic than stearic acid after prolonged exposure in differentiated HepaRG cells (unpublished data). Other investigations in liver cells reported that palmitic acid could be more cytotoxic than stearic acid (Moliterni et al., 2024; Oh et al., 2012). Second, we did not investigate the effect of maneb, mancozeb, manganese, and zinc on carbohydrate homeostasis. Indeed, alteration of glucose metabolism can also participate to the pathogenesis of fatty liver (Massart et al., 2012; Scoditti et al., 2024). Notably, there is experimental and clinical evidence linking manganese excess and zinc deficiency to the impairment of carbohydrate metabolism, in particular in the liver (Sun et al., 2023). An interesting target to study would be carbohydrate-responsive element binding protein (ChREBP), a transcription factor sensing carbohydrates and activating target genes involved in hepatic glycolysis and DNL (Massart et al., 2012; Régnier et al., 2023). For instance, regarding metal hepatotoxicity, investigations in mice and cultured HepG2 cells showed that lead (Pb²⁺) could promote DNL and induce steatosis via ChREBP activation (Vineeth Daniel et al., 2019). However, it is noteworthy that maneb, mancozeb, and manganese did not increase DNL in HepaRG cells (Supplementary Figure 5).

## 5. Conclusion

In conclusion, this study showed that maneb, mancozeb, and MnCl₂ aggravated lipid accumulation in HepaRG cells cultured with stearic and oleic acids. In contrast, chlorpyrifos, dimethoate, diazinon, iprodione,

and imazalil did not have such an effect. Moreover, low concentrations of maneb and MnCl₂ altered the expression and activity of different CYPs in +FA- and -FA-HepaRG cells, which was associated with an alteration of diazinon biotransformation. These findings could have major pathophysiological consequences in obese individuals with MASLD and exposed to the dithiocarbamates maneb and mancozeb. In addition, our investigations suggested that Zn supplementation might be an efficient strategy to prevent dithiocarbamate-induced disturbances of liver metabolism in the context of MASLD.

## Funding

This work was funded by the "Office Français de la Biodiversité" (OFB, formerly "Agence Française pour la Biodiversité"), program ECOPHYTO II", project PESTIFAT AFB/2019/87 (PR-EST, 2019-2022). Kilian Petitjean received fellowships from Cancéropole Grand-Ouest (Projet structurant PeNiCa) and OFB. Hugo Coppens-Exandier is a recipient of a fellowship "Convention Industrielle de Formation par la Recherche" (CIFRE n°221206A10) from the Association Nationale de la Recherche et de la Technologie (ANRT).

## CRediT authorship contribution statement

**Kilian Petitjean:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. **Giovanna Dicara:** Investigation, Formal analysis. **Sébastien Bristeau:** Investigation, Formal analysis. **Hugo Coppens-Exandier:** Investigation, Formal analysis. **Laurence Amalric:** Writing – review & editing, Methodology. **Nicole Baran:** Writing – review & editing, Methodology. **Camille C. Savary:** Writing – review & editing, Methodology, Conceptualization. **Anne Corlu:** Writing – review & editing, Supervision, Conceptualization. **Pascal Loyer:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Bernard Fromenty:** Writing – review & editing, Writing – original draft, Methodology, Funding acquisition, Conceptualization.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bernard Fromenty reports financial support was provided by Office Français de la Biodiversité. Kilian Petitjean reports financial support was provided by Cancéropole du Grand Ouest. Hugo Coppens-Exandier reports financial support was provided by Association Nationale de la Recherche et de la Technologie. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We are grateful to INSERM (Institut National de la Santé et de la Recherche Médicale), CNRS (Centre National de la Recherche Scientifique), and the University of Rennes for their constant administrative and financial support. We would like to thank Dr Kyle Hoehn (University of New South Wales, Sydney, Australia) for having given us the detailed protocol to investigate *de novo* lipogenesis in cultured cells. We also thank Korydwen Fruit for her assistance in some investigations.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.etap.2025.104773.
# Data availability

Data will be made available on request.

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