# A new alternative method using cyp3a65 expression in transgenic zebrafish embryos to assess metabolic endocrine-disrupting chemicals in the intestine

Chedi Erradhouani, Florian Geffroy, Benjamin Piccini, Nathalie Hinfray, Edith Chadili, Patrick Balaguer, Frédéric Sohm, Selim Aït-Aïssa, Xavier Coumoul, François Brion

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## To cite this version:

Chedi Erradhouani, Florian Geffroy, Benjamin Piccini, Nathalie Hinfray, Edith Chadili, et al. A new alternative method using cyp3a65 expression in transgenic zebrafish embryos to assess metabolic endocrine-disrupting chemicals in the intestine. Environment International, 2025, 205, pp.109872. 10.1016/j.envint.2025.109872. ineris-05330556

HAL Id: ineris-05330556

https://ineris.hal.science/ineris-05330556v1

Submitted on 3 Nov 2025

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Full length article

# A new alternative method using *cyp3a65* expression in transgenic zebrafish embryos to assess metabolic endocrine-disrupting chemicals in the intestine

Chedi Erradhouani<sup>a,b,1</sup> <sup> ID </sup>, Florian Geffroy<sup>a,b,1</sup> <sup> ID </sup>, Benjamin Piccini<sup>a</sup>, Nathalie Hinfry<sup>a ID</sup>, Edith Chadili<sup>a</sup>, Patrick Balaguer<sup>c ID</sup>, Frédéric Sohm<sup>d ID</sup>, Selim Aït-Aïssa<sup>a</sup>, Xavier Coumoul<sup>b,*</sup>, François Brion<sup>a,* ID</sup>

## ARTICLE INFO

Handling Editor: Adrian Covaci

**Keywords:**

Endocrine-disrupting chemicals

Danio rerio

In vivo bioassay

Intestine

CYP3A65

CYP3A4

## ABSTRACT

Metabolic endocrine-disrupting chemicals (MDCs) contribute to the development and increasing incidence of metabolic disorders, highlighting the need for relevant assays to identify them. Several alternative models to mammals, notably the zebrafish, have been developed, but none of these assays account for the modes of action and effects of MDCs on the intestine. This study aimed to 1) establish and characterize a transgenic embryo model expressing GFP under the control of the zebrafish *cyp3a65* gene, ortholog of the human *cyp3a4*; 2) set up an original zebrafish embryo-based bioassay to study the Effects of Metabolic Endocrine disRuptors in Gut of zebrafish Embryos (EMERGE). Spatiotemporal expression of *cyp3a65*-GFP and temporal expression of endogenous *cyp3a65* was characterized using fluorescence imaging of GFP, immunohistochemistry and RT-qPCR, under both normal and exposed conditions to clotrimazole and TCDD (respectively zfPXR and zfAhR2 agonists). Then, twenty-two chemicals were screened for their potential activity using the EMERGE assay. We report an early and dynamic expression of *cyp3a65* in the developing intestine which is disrupted in a time and concentration dependent manner by zfPXR and zfAhR2 agonists. Moreover, we report that various environmental chemicals can significantly up- or down regulate *cyp3a65* expression, most of them being newly identified as targeting the intestine and disrupt this critical metabolic enzyme. In conclusion, EMERGE represents a new easy to use assay to screen metabolic disrupting activity of chemicals in a non-mammalian model. The data collected suggest it could provide valuable insights into the effects of substances on *cyp3a4* in humans and may emerge as a valuable assay for assessing human-relevant effects.

## 1. Introduction

Numerous substances that contaminate our environment represent a growing risk to human health (Naidu et al. 2021), among which meta- bolic endocrine-disrupting chemicals (MDCs) have been identified in the onset and the rising incidence of metabolic pathologies such as obesity, type 2 diabetes and hepatic steatosis (Heindel et al. 2015; Kassotis et al. 2022). MDCs can disrupt key metabolic signaling pathways by inter- acting with nuclear and cytoplasmic receptors and the expression of proteins they regulate, leading to organ-specific effects promoting metabolic pathologies (Heindel et al. 2022). The development of tests

that can inform on the mode of action (MoA) of chemicals and their pathophysiological effects on metabolism has recently emerged, notably to fill existing gaps at the regulatory level. In that respect, the devel- opment of a panel of *in silico*, *in vitro* and *in vivo* models appears relevant for an integrative understanding of the MoAs and effects of MDCs at the molecular, cellular, organ and whole organism levels, for an effective assessment of MDCs toxicity (Bernal et al., 2022).

During the past decades, the zebrafish (*Danio rerio*) has emerged as a relevant alternative biological model to mammals for studying endo- crine disruption (Patisaul et al. 2018) because of its numerous attractive zootechnical features (e.g., inexpensive to maintain, easy to breed, fast

<footnote><sup>a</sup> Ecotoxicologie des substances et des milieux, INERIS, Verneuil-en-Halatte, France</footnote>

<footnote><sup>b</sup> Université Paris Cité, Inserm, HealthFex, F-75006 Paris, France</footnote>

<footnote><sup>c</sup> Inserm U1194, Montpellier, France</footnote>

<footnote><sup>d</sup> INRAE USC 1370, ENS, CNRS UMR 5242, Université Claude, Bernard-Lyon 1, Lyon, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: xavier.coumoul@u-paris.fr (X. Coumoul), francois.brion@ineris.fr (F. Brion).</footnote>

<footnote n="1">¹ These two authors contributed equally to this work.</footnote>
development) (Lieschke and Currie 2007), genetic and anatomical
proximity to humans (e.g., 71% of the human protein-coding genes have
zebrafish orthologs) (Howe et al. 2013; Sassen and Köster 2015), and
gene-editing technologies allowing the development of mutant and
transgenic lines (e.g., Tol2 transposon system), which significantly
stimulated research on zebrafish (Choi et al. 2021). As a versatile model,
the zebrafish embryo allows high-content and extensive screening of
pharmaceuticals and chemicals (Wang et al. 2025; Wiley et al. 2017).
This is highly relevant for biomedical and environmental research, as
well as hazard characterization of chemicals, particularly in a regulatory
context supporting toxicity and endocrine activity assessment through
OECD test guidelines (e.g., OECD TG No. 236 and No. 250). In the
context of metabolic toxicity, the zebrafish model has been increasingly
used, and several studies have demonstrated that chemical exposures
can lead to metabolic disruption, including effects on adipogenesis,
metabolic signaling, metabolic rate, food intake and yolk lipid absorp-
tion (Boom et al. 2024; Den Broeder et al. 2017; Martínez et al. 2020;
Mentor et al. 2020; Ortiz-Villanueva et al. 2017; Tu et al. 2019). These
studies support the relevance of the zebrafish model to investigate
metabolic disruption. Based on these findings, specific zebrafish bio-
assays have been developed mainly to investigate potential effects on
liver steatosis and metabolic rate in embryos and adipogenesis in juve-
niles (Audouze et al. 2020; Heusinkveld et al. 2024; Le Mentec et al.
2023; Renquist et al. 2013; Tingaud-Sequeira et al. 2011). However, to
our knowledge, no specific bioassays investigating the MoAs and/or
effects of MDCs in the intestine have been developed up until now. While
this organ has pleiotropic physiological functions as in humans, it is
generally less investigated in this context compared to other metabolic
organs such as the liver or the adipose tissue, calling for the development
of models to account for the effects of MDCs on intestinal metabolism
 Erradhouani et al. 2024). Moreover, the zebrafish appears as a relevant
alternative vertebrate model in order to investigate the effects of MDCs
in the gastrointestinal tract (Erradhouani et al. 2024).

Among the potential relevant molecular and biochemical targets of MDCs, the human cytochrome P450 3A4 (CYP3A4) is predominantly expressed in the liver and the proximal part of the intestine, where it represents 78 % of the total CYP450s isoforms expressed in the human small intestine (Grangeon et al. 2021; Paine et al. 2006). CYP3A65, a zebrafish ortholog of the human CYP3A4, is encoded by the *cyp3a65* gene which is 54 % identical to human *cyp3a4* (Goldstone et al. 2010) and mainly expressed in the liver and the foregut in both adults and larvae (Bainy et al. 2013; Chang et al. 2013; Tseng et al. 2005). In humans, CYP3A4 is involved in both endogenous and xenobiotic metabolism (Chen et al. 2014; van Herwaarden et al. 2007). It is regulated by several transcription factors such as the pregnane X receptor (PXR) which plays a direct role in its expression (Zhou et al. 2009), the aryl hydrocarbon receptor (AhR) which plays an indirect role by promoting PXR expression (Rasmussen et al. 2017), and the glucocorticoid receptor (GR), which directly regulates CYP3A4 expression and can also enhance the expression of PXR (Matsunaga et al. 2012; Cooper et al. 2008). In zebrafish, both ligand-activated zfPXR (e.g., clotrimazole, pregnenolone) and ligand-activated zdAhR2 (e.g., TCDD, PCB126) upregulate *cyp3a65* expression, these two activated signaling pathways interacting in a complex manner (Chang et al. 2013; Kubota et al. 2015; Erradhouani et al. 2025). Finally, we have recently shown that the zebrafish GR (zfGR) also plays a role in regulating this gene in zebrafish embryos, recalling human CYP3A4 regulation (Erradhouani et al. 2025).

Although the role of CYP3A65 in testosterone and 17β-estradiol
hydroxylation has been demonstrated in the liver, similar to the function
of CYP3A4 in humans (Chng et al. 2012; Scornaienchi et al. 2010), in-
formation on its enzymatic activity is limited. However, in various
salmonid intestinal and liver cellular models, a recent study has shown
that CYP3A-like enzymes play a role in phase 1 biotransformation of
exogenous compounds such as pharmaceuticals and micropollutants
(Martin et al. 2025), reflecting the role of CYP3A4 as a major human
metabolizing enzyme in the liver and intestine.

In the present study, we developed a transgenic zebrafish line
expressing the Green Fluorescent Protein (GFP) under the control of the
zebrafish *cyp3a65* promoter with the view to build a new biological tool
to investigate the effect of chemicals on intestinal metabolism. In order
to characterize the transgenic line, we investigated *cyp3a65* expression
during embryo development, from 24 to 120 h post fertilization (hpf), by
*in vivo* 2D fluorescence imaging. In addition, we compared these results
with measurements of endogenous *cyp3a65* expression by RT-qPCR.
Furthermore, we assessed the time- and concentration-dependent ef-
fects of literature-reported ligands of zfPXR (Creusot et al. 2021) and
zfAhR2 receptors (Bugel et al. 2013) on both GFP and *cyp3a65* expres-
sion during embryonic development.

This thorough characterization led to define robust experimental
conditions enabling the use of this transgenic line for chemical
screening. We established a new zebrafish embryo-based assay that uses
tg(cyp3a65:GFP) zebrafish to investigate the effect of MDCs in the in-
testine at eleuthero-embryonic life stages. This assay, called EMERGE
(Effects of Metabolic Endocrine disRuptors in Gut of zebrafish Embryos),
was developed within the European OBERON H2020 project which
intended to develop an integrated approach to detect endocrine
disruptors-related metabolic disorders (European Commission 2019). In
this context, EMERGE was used to screen chemicals selected by the
OBERON H2020 project. These substances were chosen for their wide-
spread use, potential toxicity and the fact that they are of regulatory
interest (bisphenol derivatives, di(2-ethylhexyl)phthalate, cadmium
chloride, perfluorooctanoic acid, 4-4'-dichlorodiphenyldichloro-
ethylene) (Audouze et al. 2020). In addition, we selected other chem-
icals on the basis of their ability to induce metabolic disorders
(chlorpyrifos, tributyltin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, triclo-
san, amiodarone chloride) (Heindel et al. 2022; Anthérieu et al. 2011),
because they are substances of concern (the polycyclic aromatic hy-
drocarbons (PAHs) 5,12-naphthacenequinone and benz[a]anthracene-
1,12-dione) or because they have been used as ligands in our previous
work to highlight the signaling pathways involved in *cyp3a65* regula-
tion, in addition to being emerging pollutants (1,2,3,7,8-penta-
chlorodibenzo-p-dioxin, clobetasol propionate, clotrimazole,
desogestrel, fluticasone propionate) (Erradhouani et al. 2025). By
screening substances from various chemical families, with a wide range
of structural diversity (SM Table S1), we aimed to assess the potential of
this approach to inform if environmental chemicals target the intestine
and modulate *cyp3a65* gene expression in a developing vertebrate
model, shedding light on their potential role in metabolic disorders.

## 2. Methods

2.1. Chemicals

1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 4,4'-dichlor-
odiphenyldichloroethylene (4,4'-DDE), 5,12-Naphthacenequinone
(5,12-NQ), amiodarone hydrochloride (AMD), bisphenol A (BPA),
bisphenol B (BPB), bisphenol C (BPC), bisphenol F (BPF), bisphenol S
(BPS), cadmium chloride (CdCl₂), clotrimazole (CLO), chlorpyrifos
(CPF), di(2-ethylhexyl)phthalate (DEHP), desogestrel (DGT), fluticasone
propionate (FLU), perfluorooctanoic acid (PFOA), tributyltin (TBT) and
triclosan (TCS) were obtained from Sigma-Aldrich (St Quentin Fallavier,
France); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from LGC Stan-
dards (Molsheim, France); benz[a]anthracene-1,12-dione (BaAQ) from
Cil Cluzeau (Sainte-Foy-la-Grande, France); clobetasol propionate
(CLOBE) from USP Ref Standard (Frederick, USA); and tetrachlorobi-
sphenol A (TCBPA) from ABCR (Karlsruhe, Germany). Chemicals were
dissolved in dimethyl sulfoxide (DMSO) for stock solutions and stored at
-20 °C. CAS numbers are indicated in supplementary material (SM Table
S1).
## 2.2. Generation of transgenic tg(cyp3a65:GFP) in transparent Casper mutant zebrafish

The transgenic line was established using Casper (roy^−/−, nacre^−/−) mutants that completely lack melanocytes and iridophores facilitating *in vivo* observations through teguments (White et al. 2008). Tol2 transposon transgenesis protocol was performed according to Kawakami et al. (2004). About 1 nL of a DNA/RNA solution containing 15 ng/µL circular DNA of vector pEXP24-21_cyp3a65_5,4kb and 8 ng/µL transposase mRNA were injected into 1 cell stage fertilized eggs. This plasmid contains two reporter genes: one coding for the GFP under the control of zebrafish *cyp3a65*, the target gene of this model, and the second one coding for Cyan Fluorescent Protein (CFP) under the control of *β-crysstallin* to identify and select transgenic individuals based on the fluorescence of their eyes, even at early stage (24 hpf). Positive F0 embryos were selected under an epi-fluorescence binocular and raised until adulthood before being crossbred with non-transgenic Casper zebrafish. The founders transmitting the transgene to 50 % of the progeny were then selected to give rise to the new generation by crossing them with non-transgenic Casper zebrafish. The same procedure was then repeated for each new generation up to F6 where the transgenic fish are crossed with each other in order to obtain 100 % transgenic homozygous embryos and maximize the number of individuals available per experiment. Currently, the ninth generation (F9) is housed in our breeding unit.

## 2.3. Animal facilities

Fish were reared in 3.5 L aquaria at the INERIS (Verneuil-en-Halatte, France) animal facilities, alternating 14 h of light and 10 h of darkness with a water recirculation system (Zebtec, Tecniplast, Italy). Temperature (27.0 ± 2.0 °C), pH (8.1 ± 0.1), conductivity (370 ± 20 µS/cm), dissolved oxygen (> 90 %), nitrate (not detectable), nitrite (not detectable) and ammonia (not detectable) were monitored in water every day. Fish were fed with flake food (SDS400, Scientific Fish Food) in the morning and newly hatched brine shrimp (Sep-Art, Ocean Nutrition Europe) in the afternoon. Transgenic pairs of adult fish (1 female and 1 male) were formed the day before egg collection. Fertilized eggs were sorted the following morning, then placed in water at 28.0 ± 1.0 °C with the same light/dark cycle (14 h/10 h).

## 2.4. Tg(cyp3a65:GFP) embryo exposures

Tg(cyp3a65:GFP) zebrafish embryos were exposed during 48 h from day 3 to 5 post-fertilization (dpf) to chemicals at sublethal concentrations (i.e., that do not elicit toxicity (> 10 % mortality) or developmental effect that could preclude a non-specific measurement). All tested concentrations are listed in *Supplementary Material* (SM). Depending on the chemical tested, between one and six independent experiments were performed. The solvent DMSO (0.01 % v/v) is used as solvent control and clotrimazole 1.5 µM or TCDD 0.05 nM were used as positive control for each experiment. Each exposure group includes a total of 21 embryos distributed in 3 crystallizing dishes containing 7 embryos each. Chemicals were dissolved in breeding water with 2 mL of contaminated water per embryo (i.e., 14 mL per crystallizing dish). Embryos were kept in an incubator at 28.0 ± 1.0 °C with a 14 h light/10 h dark cycle, under semi-static conditions and a renewal of contaminated water after 24 h, which corresponds to 96 hpf. Exposure to zfPXR and zfAhR2 agonists (clotrimazole and TCDD respectively) follows the same specifications.

## 2.5. In vivo fluorescent imaging

After 48 h of exposure, at 120 hpf, living *tg(cyp3a65:GFP)* embryos were observed individually in lateral view to detect intestinal fluorescence. Images were captured using a Zeiss AxioImager.Z1 fluorescence microscope with an AxioCam Mrm camera (Zeiss GmbH, Göttingen, Germany) and a 10 ms exposure time using a X10 objective. ImageJ

software was used for fluorescence quantification with a macro-control developed at the laboratory (Schindelin et al. 2012). For each embryo, the grey values of all the pixels in the intestinal part (region of interest) are summed. A grey-value detection threshold of 159 was fixed to avoid fish autofluorescence, based on non-transgenic embryo fluorescence. Details can be found in supplementary material file named *determination of fluorescence threshold grey-value*. Each image was checked individually and processed manually to exclude, when necessary, non-specific fluorescence and define specific regions of interest to analyze.

## 2.6. Quantification of selected genes expression:zfahr2, zfpxr and cyp3a65

Real-time quantitative PCR (RT-qPCR) was conducted to evaluate the mRNA expression levels of endogenous genes involved in the regulation of CYP3A65 (*zfahr2*, *zfpxr* and *cyp3a65*). Following *in vivo* fluorescence imaging, embryos were immediately pooled (n = 5 embryos per pool and 4 pools per condition), transferred to RNA stabilization solution (RNAlater™) and stored at -80 °C to preserve the samples for subsequent RNA extraction. RNA extraction was performed with MAX-MAX™ *mirVana™* Total RNA Isolation Kit (ThermoFisher Scientific, USA) after whole embryo grinding with 1 mm glass beads (2 x 10 sec, speed: 7.5 m/sec) with a FastPrep-24™ 5G (MP Biomedicals, France). Extracted RNA quantity and purity (260/280 ratio) were assessed using a NanoDrop™ 8000 spectrophotometer (ThermoFisher Scientific, USA), prior to storage at -80 °C for further analysis. Then, 200 ng of total RNA were used for cDNA synthesis using Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific, USA). The reaction mixture was incubated at 50 °C for 30 min, then heated at 85 °C for 5 min to stop the reaction.

Primers for *zfahr2* and *zfpxr* were sourced directly from the manufacturer (ThermoFisher, USA), references are provided in SM Table S2. Since commercial primers for *cyp3a65* were unavailable, custom primers were designed by the same supplier (SM Table S2). Quantitative PCR (qPCR) was performed with the LightCycler® 96 system (Roche Diagnostics, France) using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, USA). The thermal cycling program began with a UNG (uracil-N-glycosylase) activation step at 50 °C for 2 min to eliminate potential genomic DNA contamination, followed by enzyme activation (polymerase) at 95 °C for 20 sec. Amplification consisted of 40 cycles, each including denaturation at 95 °C for 10 sec and hybridization/elongation at 60 °C for 30 sec. This was followed by a final cooling step at 37 °C for 900 sec. Absolute quantification of target gene expression was carried out using a standard curve composed of 15 serial dilutions.

## 2.7. GFP fluorescence immunohistochemistry

Tg(cyp3a65:GFP) embryos were fixed in modified Davidson's fixative (22 % v/v formaldehyde 37-40%; 11.5 % v/v glacial acetic acid; 33 % v/v ethyl alcohol 95%; 33.5 % v/v distilled water) for 48 h at room temperature. After fixation, samples were dehydrated in ethanol, cleared in isopropanol and paraffin embedded. Embryos were then sectioned at 5 µm and immunohistochemistry was performed using InsituPro VSI station (Intavis Inc., Köln, Germany) according to the protocol described in Hinfry et al. (2013). Monoclonal mouse-produced anti-GFP primary antibody diluted at 1:500 (Abcam, Paris, France, reference number ab38689) was used to detect GFP in *situ*.

## 2.8. Data analysis and statistics

For each experiment, data from individual embryo GFP fluorescence value or mRNA for pooled individuals are normalized as fold-change (compared to mean of solvent control). Data are expressed as violin plots to account for data distribution, with median and quartiles. Depending on the structure of the data, parametric or non-parametric tests were used for statistical analysis on GraphPad Prism 8.0.1: Mann-
Whitney *U* test or *t* test for single-condition experiments and ordinary one-way ANOVA with Tukey's/Dunnett's post-hoc or Kruskal-Wallis with Dunn's post-hoc for multiple comparison. For concentration-response experiments, outliers (detected with GraphPad Prism: ROUT method with Q = 1 %) were removed before statistical tests when

detected. Stars represent p-values: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****). For each substance tested, the first concentration producing a statistically significant difference from control was defined as the LOEC (Lowest Observed Effect Concentration), and the lowest value across LOECs from independent experiments was

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Fluorescence expression in developing zebrafish embryos. (A) Optical microscopy images of zebrafish embryos from 4 to 120 hpf in breeding water. (B) GFP immunohistochemistry staining at 120 hpf showing localization in the anterior part of the intestine in control condition (green) and nucleus DNA staining (blue). Blue arrow shows CFP fluorescence in the eye, green arrow shows GFP fluorescence in the intestine. Pictures are reworked in terms of contrast to highlight the GFP fluorescence signal over time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
selected. In addition, for each experiment, the maximal fold-change was determined at the concentration producing the strongest effect, and the median of this value across independent experiments was calculated for each substance.

# 3. Results

## 3.1. Localization of the GFP in developing embryos

The expression pattern of the GFP was investigated in developing *tg(cyp3a65:GFP)* embryos from 4 to 120 hpf using *in vivo* imaging over time and immunohistochemistry staining at 120 hpf. GFP expression was detectable as from 72 hpf in some individuals (Figs. 1A and 2A) and was localized in the anterior part of the intestine (Fig. 1B).

## 3.2. Comparison of GFP and endogenous cyp3a65 expression profiles at early-stage development

As observed with GFP, the kinetics of *cyp3a65* mRNA expression monitored from 24 hpf to 120 hpf showed a starting expression at 72 hpf (Fig. 2B), which is in line with previous studies (Nawaji et al. 2020; Tseng et al. 2005). Interestingly, intestinal development in zebrafish starts from 18 hpf and is in its major part completed at 120 hpf, performing most of intestinal functions (Zhao and Pack 2017). Monitoring of CYP3A65 protein was not possible in the absence of specific antibodies raised against zebrafish CYP3A65. However, considering the temporal expression of the GFP reporter gene, the fluorescent signal in the intestine starts at 72 hpf, as it is detected in some individuals, and then quantified in most individuals at 96 hpf (Fig. 2A). Moreover, the quantification of GFP until 120 hpf (Fig. 2A) is strongly associated with

the *cyp3a65* mRNA expression measured by RT-qPCR (Fig. 2B), both endpoints sharing a similar profile with a significant increase in their expression at 96 hpf which continues up to 120 hpf. The temporal expression and the profile of GFP fluorescence and *cyp3a65* mRNA support that GFP mimics the temporal expression of the endogenous gene.

## 3.3. Dynamic of expression of zfahr2 and zfpxr during embryonic development

We further investigated the expression of *zfpxr* and *zfahr2* genes as putative response elements for these two ligand-activated transcriptional factors were identified in the promoter region of *cyp3a65* (Erradhouani et al. 2025), and literature data report their involvement in its regulation by using agonists such as dexamethasone and TCDD (Chang et al. 2013; Kubota et al. 2015). Interestingly, the gene expression of *zfpxr* and *zfahr2* is strongly correlated to *cyp3a65* expression profile through time (Figs. 2C and 2D), showing an increase during development up to 120 hpf. These two ligand-activated transcription factors are indeed expressed very early during zebrafish development (Eide et al. 2021), reflecting the establishment of the molecular players involved in *cyp3a65* expression.

## 3.4. Concentration-response of cyp3a65 in zebrafish embryos exposed to zfpXR and zfAhR2 agonists

Both zfpXR and zfAhR2 agonists, i.e., clotrimazole and TCDD, induce the GFP expression in a concentration-dependent manner, as shown in Figs. 3A and 3D, indicating that both zfpXR and zfAhR2 ligand-activated transcription factors are involved in the up-regulation of *cyp3a65*

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** GFP fluorescence intensity in *tg(cyp3a65:GFP)* embryos and mRNA expression of *cyp3a65*, *zfahr2* and *zfpxr* during embryonic development (A) GFP intensity in individual embryos (n = 21 embryos per condition minimum) from 24 to 120 hpf in control condition (DMSO 0.01 %) and mRNA expression of (B) *cyp3a65*, (C) *zfahr2* and (D) *zfpxr* in pooled zebrafish embryos (n = 5 embryos/pool) in same condition. a, b, c: conditions sharing the same letter are not statistically different (p < 0.05). Red dotted line (A) indicates individuals under the detection limit.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effect of CLO and TCDD on GFP fluorescence intensity and *cyp3a65* mRNA expression. GFP intensity in individual embryos (n = 21 iembryos per condition minimum) at 120 hpf after 48 h exposure to (A) CLO and (D) TCDD (control: DMSO 0.01 %) and *cyp3a65* mRNA expression in pooled zebrafish embryos (n = 5 embryos/pool) in (B) CLO and (E) TCDD. GFP immunohistochemistry staining (green) in the intestine after 48 h exposure to (C) CLO 1.5 µM and (F) TCDD 0.05 nM; blue: nucleus DNA staining). Green arrow shows GFP fluorescence in the intestine. Pictures were reworked in terms of contrast to highlight the GFP fluorescence signal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

expression. Moreover, parallel expression of *cyp3a65* mRNA was also observed (Figs. 3B and 3E), confirming that GFP fluorescence is consistent with endogenous *cyp3a65* mRNA expression.

In order to assess whether GFP expression remains localized in the intestine under chemical exposure conditions, immunohistochemistry staining was performed at 120 hpf and confirmed its localization in the foregut (Figs. 3C and 3F). Moreover, GFP immunochemistry expression after CLO and TCDD exposure is higher and more extended than in the control condition (Fig. 1B).

Altogether, these results demonstrate that both zfPXR and zfAhR2 agonists (clotrimazole and TCDD) regulate *cyp3a65* expression in a similar manner, with GFP fluorescence faithfully reflecting the endogenous mRNA expression and remaining localized in the foregut.

### 3.5. Time-dependent effect of CLO and TCDD on GFP intensity

To get insights into the temporal GFP expression after exposure to chemicals, we monitored GFP expression at 72, 96 and 120 hpf in embryos exposed to either CLO or TCDD starting at 72 hpf. The results from these kinetics showed an increase in GFP intensity over time, with a significantly different induction from the control within 24 h of treatment for both exposure conditions (Figs. 4A and 4B). Thus, according to our protocol, CLO and TCDD activate the transgene significantly after only 24 h of exposure.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Time-dependent effect of CLO and TCDD on GFP fluorescence intensity. GFP intensity was monitored in developing embryos (n = 24) exposed to (A) clotrimazole (CLO) 1.5 µM or (B) TCDD 0.05 nM from 72 hpf and observed at 72; 96 and 120 hpf. Same individuals are observed for each time point. Control condition corresponds to DMSO 0.01 %.</figcaption></figure>
## 4. Establishment of the EMERGE assay

To evaluate the *tg(cyp3a65:GFP)* transgenic line potential to become an efficient new approach methodology for metabolic disruption assessment, we developed the EMERGE assay (Effect of Metabolic Endocrine disruptors in Gut of zebrafish Embryos) by designing a single and well-defined protocol for chemical screening. This protocol, illustrated in Fig. 5, follows the experimental steps of existing OECD test guidelines using zebrafish transgenic lines, such as the EASZY assay, which uses the *tg(cyp19a1b:GFP)* transgenic line to assess estrogenic activity in the brain (Brion et al. 2012; OECD Test No. 250: EASZY assay 2021).

Based on the results shown in Fig. 3, we selected DMSO 0.01 % as the solvent control and CLO 1.5 µM or TCDD 0.05 nM as positive controls. Figs. 1 and 2 showed that *cyp3a65* and GFP expression begin at 72 hpf, while Fig. 4 suggested a stronger induction at 120 hpf. From these observations, we selected a 48 h exposure period, from 72 hpf to 120 hpf, with a contaminated water renewal at 24 h (96 hpf). The number of individual embryos and the image analysis protocol were selected based on the OECD Test No. 250 guideline.

In order to test the EMERGE assay and evaluate its ability to detect MDCs, 22 chemical substances from various families were screened at different sublethal concentrations using this well-defined protocol (Fig. 5).

### 4.1. Screening of MDCs in EMERGE assay

Firstly, the screening of the 22 selected chemical substances confirmed the localization of *cyp3a65* expression in the gut of transgenic zebrafish embryos after exposure to chemicals, and not in other tissues (e.g., liver). As with exposure to CLO and TCDD (Fig. 3), fluorescence remains located in the foregut (Fig. 6). However, the extent of the fluorescent area throughout the digestive tract varied depending on the chemical, from the foregut to the anus for the most inductive substances (e.g., TCDD, TCS) (Fig. 6).

The results of the screening reveal that many chemicals were capable

of statistically significantly modulate the expression of intestinal *cyp3a65*, either by inducing (n = 14) or by inhibiting (n = 4) GFP expression (Table 1). Only 4 out of 22 chemicals were totally inactive (BPS, TCBPA, TBT and DEHP).

Among these effects, differences in activity can be observed between the substances tested, with LOECs varying widely depending on the chemical family, from the pM to the µM range (Fig. 7 and Table 1).

For each substance, between one to six independent experiments were performed. Complete datasets from all independent experiments are available in the Supplementary Material (SM Figs. S1–S22), and one representative concentration-response experiment is illustrated below.

### 4.2. Dioxins and PAHs

In EMERGE assay, dioxins and PAHs were found among the most potent inducers of *cyp3a65* expression (Fig. 7 and Table 1). TCDD and PeCDD had the highest biological activity of all the 22 chemicals screened, showing a significant induction of fluorescence at very low concentrations, with LOECs in the pM range (TCDD: 5 pM; PeCDD: 2.7 pM). PeCDD consistently induced GFP at all tested concentrations (Fig. 8B). TCDD displayed a concentration-dependent response (Fig. 8C), with a maximal median 6.33-fold induction over control at 0.05 nM.

For PAHs, both BaAQ and 5,12-NQ significantly increased intestinal GFP fluorescence at low concentrations, from the lowest concentration tested (5,12-NQ: 0.27 µM, BaAQ: 0.04 µM) up to the highest tested concentrations (5,12-NQ: 2.5 µM, BaAQ: 0.37 µM) (Figs. 8D and 8E).

### 4.3. Bisphenols

Among the six bisphenols tested, BPA, BPB, BPC and BPF significantly induced intestinal GFP fluorescence, whereas no significant effect was measured after exposure to BPS and TCBPA.

BPB, BPC and BPF all showed concentration-dependent induction of GFP (Figs 9A, 9B and 9C), from LOECs in the µM range (BPB: 10.5 µM; BPC: 7.5 µM; BPF: 12.5 µM) up to the highest concentrations tested (BPB:

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Graphical overview of the EMERGE assay. *Tg(cyp3a65:GFP)* zebrafish embryos are exposed to chemicals during 48 h, from 72 to 120 hpf. Then, individual *in vivo* imaging of each embryo is performed to quantify the GFP fluorescence before analysis in ImageJ Fiji image processing program and data analysis.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** GFP fluorescence expression in *tg(cyp3a65:GFP)* embryos at 120 hpf exposed 48 h to various MDCs in the EMERGE assay. Photos represent one individual intestine for the 22 MDCs screened in EMERGE at high concentrations.</figcaption></figure>
<table><caption>**Table 1**
Activity profiles, LOECs, and maximal fold-changes of all compounds tested in the EMERGE assay.</caption><thead><tr><th rowspan="2">Family</th><th rowspan="2">Chemicals</th><th colspan="2">Tested concentration (µM)</th><th colspan="6">Biological activity in independent EMERGE assays</th><th rowspan="2">LOEC (µM)</th><th rowspan="2">Median maximal fold-change</th></tr><tr><th>Min</th><th>Max</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th></tr></thead><tbody><tr><td rowspan="2">Dioxins</td><td>PeCDD</td><td>2.70E-06</td><td>2.50E-05</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td></td><td></td><td>2.70E-06</td><td>2.3</td></tr><tr><td>TCDD</td><td>1.70E-06</td><td>1.50E-04</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>5.00E-06</td><td>6.3</td></tr><tr><td rowspan="2">Oxy-PAHs</td><td>5,12-NQ</td><td>0.27</td><td>2.5</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>0.27</td><td>3.0</td></tr><tr><td>BaAQ</td><td>0.04</td><td>0.37</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>0.04</td><td>4.0</td></tr><tr><td rowspan="6">Bisphenols</td><td>BPA</td><td>1.9</td><td>30.4</td><td>✓</td><td>✓</td><td>n.e</td><td>✓</td><td></td><td></td><td>21.9</td><td>1.9*</td></tr><tr><td>BPB</td><td>3.5</td><td>14</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td></td><td></td><td>10.5</td><td>3.3</td></tr><tr><td>BPC</td><td>2.5</td><td>10</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td></td><td></td><td>7.46</td><td>3.0</td></tr><tr><td>BPF</td><td>3.12</td><td>100</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td></td><td></td><td>12.5</td><td>4.2</td></tr><tr><td>BPS</td><td>11</td><td>100</td><td>n.e</td><td>n.e</td><td></td><td></td><td></td><td></td><td>n.e</td><td>n.e</td></tr><tr><td>TCBPA</td><td>0.3</td><td>1.2</td><td>n.e</td><td>n.e</td><td></td><td></td><td></td><td></td><td>n.e</td><td>n.e</td></tr><tr><td rowspan="4">Biocides/Pesticides</td><td>CPF</td><td>0.3</td><td>1</td><td>✓</td><td></td><td>n.e</td><td>✓</td><td></td><td></td><td>0.3</td><td>3.29</td></tr><tr><td>DDE</td><td>0.03</td><td>1</td><td>✓</td><td></td><td>n.e</td><td>✓</td><td></td><td></td><td>0.1</td><td>0.075*</td></tr><tr><td>TBT</td><td>0.034</td><td>170</td><td>n.e</td><td>n.e</td><td></td><td></td><td></td><td></td><td>n.e</td><td>n.e</td></tr><tr><td>TCS</td><td>0.031</td><td>0.864</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>0.093</td><td>4.3</td></tr><tr><td rowspan="6">Pharmaceuticals</td><td>AMD</td><td>0.1</td><td>1</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>0.3</td><td>3.4</td></tr><tr><td>CLO</td><td>0.019</td><td>1.5</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>0.17</td><td>5.2</td></tr><tr><td>DGT</td><td>0.11</td><td>1</td><td>✓</td><td>✓</td><td>n.e</td><td>n.e</td><td>n.e</td><td>n.e</td><td>1</td><td>3.0*</td></tr><tr><td>CLOBE</td><td>9.5E-04</td><td>0.24</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>0.061</td><td>0.047</td></tr><tr><td>FLU</td><td>2.7E-03</td><td>0.225</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>0.025</td><td>0.060</td></tr><tr><td>CdCl<sub>2</sub></td><td>0.1</td><td>1</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td></td><td>1</td><td>0.15</td></tr><tr><td>Metals</td><td>DEHP</td><td>1</td><td>30</td><td>n.e</td><td>n.e</td><td></td><td></td><td></td><td></td><td>n.e</td><td>n.e</td></tr><tr><td>Perfluorinated compounds</td><td>PFOA</td><td>1</td><td>241.5</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td></td><td></td><td>12.5</td><td>1.98</td></tr></tbody></table>

This table summarizes, for all compounds tested in EMERGE, their chemical family and the range of tested concentrations. 15 to 21 tg(cyp3a65:GFP) zebrafish embryos were analyzed per concentration tested in each experiments, and fluorescence data were normalized to the solvent control and expressed as fold-change (treated/control) before statistical analysis. Compound activity is reported for each independent experiments (n.e = no effect, ✓ = statistically significant induction, ✓ = statistically significant inhibition). The LOEC reported is the lowest value across all independent experiments. For each compound, the median and range (min-max) of the maximal fold-changes observed across all experiments are detailed. *For substances showing different effects across independent experiments (i.e BPA, DDE, DGT), only experiments with a statistically significant activity were considered to calculate the median maximal fold-change.

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Active chemicals by LOEC in EMERGE. Orange triangles represent *cyp3a65* inductors and blue triangles represent *cyp3a65* inhibitors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

14 µM; BPC: 10 µM; BPF: 100 µM). The maximal measured induction of GFP as compared to control varied from 3-fold (BPC) to 4.2-fold (BPF) (Table 1).

BPA also induced intestinal GFP expression. However, the effect was less pronounced (median maximal fold-change of 1.88) as compared to its alternatives. Significant induction was observed in three out of four experiments, only at the highest concentrations tested (21.9 or 30.4 µM depending on the experiment) (SM Fig. S5).

### 4.4. Biocides/Pesticides

Chlorpyrifos (CPF) and triclosan (TCS) both significantly induced intestinal fluorescence (Figs. 10A and 10C), with median measured maximal fold-inductions of 3.29 and 4.32 respectively. Those inductions

were measured at low concentrations (LOECs of 0.3 µM for CPF and 0.093 µM for TCS) up to the highest concentration tested (1 µM for CPF and 0.864 µM for TCS).

4,4'-dichlorodiphenyldichloroethylene (4,4'-DDE) was able to significantly inhibit GFP expression. This inhibitory effect was observed in two out of three independent experiments, with LOECs varying from 0.1 to 1 µM depending on the experiment, with a fluorescence extinction observed at the highest concentration tested (1 µM) (SM Fig. S12).

Finally, no statistically significant change in GFP expression was observed after exposure to tributyltin (TBT) in two independent experiments (Fig. 10B).
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Representative concentration-responses obtained in EMERGE for dioxins (B-C) and oxy-PAHs (D-E). GFP expression in zebrafish embryos exposed to (A) dimethylsulfoxide (DMSO); (B) 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD); (C) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); (D) 5,12-Naphtacenequinone (5,12-NQ); (E) benz(a)anthracene-1,12-dione (BaAQ). \* denotes significant differences as compared to control DMSO 0.01 \%; Kruskal Wallis test followed by Dunn's test; P ≤ 0.05 (\*), P ≤ 0.01 (\*\*\*) and P ≤ 0.0001 (\*\*\*\*). Data are represented as violon plot with each point representing fluorescent intensity in individual embryo, expressed as fold GFP intensity above control; solid lines represent the median of each test condition. Outliers (detected with GraphPad Prism: ROUT method with Q = 1 \%) were removed. All independent experiments can be found in supplementary material.</figcaption></figure>

## 4.5. Pharmaceuticals

Among the pharmaceuticals tested in EMERGE, clotrimazole (CLO), an azole fungicide, significantly induced GFP fluorescence (Fig. 11B). In all experiments, a strong induction (median 5.15-fold over control) was measured at the highest concentration tested of 1.5 µM, while in some experiments, a significant induction of intestinal GFP was observed at lowest concentrations (LOEC of 0.17 µM) (SM Fig. S16).

Desogestrel (DGT), a synthetic progestin, was found to be active in EMERGE in two experiments, as shown by its ability to induce GFP with a maximal 3-fold induction at 1 µM. However, this effect is not consistently reported among the six independent experiments (SM Fig. S17 and Table 1).

Amiodarone (AMD), an antiarrhythmic pharmaceutical, also significantly induced intestinal fluorescence in two independent experiments, at 0.3 and 1 µM, with a median measured fold-induction of 3.43 over control at 1 µM.

Among the pharmaceuticals tested, two synthetic glucocorticoids (fluticasone propionate (FLU) and clobetasol propionate (CLOBE)) showed a concentration-dependent effect on GFP fluorescence, with a significant inhibition from 25 nM for FLU and 61 nM for CLOBE up to a complete extinction of the fluorescence signal at the highest concentrations tested (225 nM for FLU and 243 nM for CLOBE) (Figs. 11C and 11D).

## 4.6. Others

The heavy metal cadmium chloride (CdCl₂) significantly inhibited GFP expression (median 0.147-fold over control), but only at the highest concentration tested (1 µM) (Fig. 12A).

The di(2-ethylhexyl)phthalate (DEHP) did not elicit any significant change in intestinal fluorescence at the concentrations tested (Fig. 12B).

The perfluorooctanoic acid (PFOA) was found to be active in EMERGE as shown by its capacity to induce GFP fluorescence in all
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Representative concentration-responses obtained in EMERGE for bisphenols. GFP expression in zebrafish embryos exposed to (A) bisphenol B (BPB); (B) bisphenol C (BPC); (C) bisphenol F (BPF); (D) bisphenol S (BPS); (E) tetrachlorobisphenol A (TCBPA). \* denotes significant differences as compared to control DMSO 0.01%; Kruskal Wallis test followed by Dunn's test; $P \le 0.01$ (\*\*), $P \le 0.001$ (\*\*\*\*) and $P \le 0.0001$ (\*\*\*\*\*). Data are represented as violon plot with each point representing fluorescent intensity in individual embryo, expressed as fold GFP intensity above control; solid lines represent the median of each test condition. Outliers (detected with GraphPad Prism: ROUT method with $Q = 1\%$) were removed. BPA is not represented as results varied across experiments. All independent experiments can be found in supplementary material.</figcaption></figure>

experiments ($n = 4$). However, the LOEC varied across independent experiments (between 12.5 and 120 µM) (SM Fig. S22). This compound induced GFP with a median maximal 2-fold over control.

# 5. Discussion

## 5.1. Strengths and limits of the EMERGE assay

The established EMERGE assay, based on the *tg(cyp3a65:GFP)* transgenic line, represents a new integrative assay to screen potential MDCs. Zebrafish embryos remain alive during fluorescence imaging under the microscope, and efforts are made to minimize manipulations, thereby reducing stress and safeguarding their well-being. Individual GFP measurements offer a significant advantage over RT-qPCR gene quantification which is carried out on pools of 5 embryos (to obtain sufficient biological material). In contrast, the *tg(cyp3a65:GFP)* model allows assessment of *cyp3a65* expression in a single individual, with potential repeated observations on the same embryo over time.

The exclusively intestinal localization of *cyp3a65* expression in EMERGE is of great interest, as the effects of MDCs are poorly

investigated in the intestine compared to liver or adipose tissue (Erradhouani et al. 2024). The dynamics of its expression indicate the gradual establishment of the biochemical material required for CYP expression and, more generally, highlights the development of the gastrointestinal tract as the first MDCs barrier. As the morphogenesis of the digestive tract is completed around 34 hpf, the significant increase in intestinal size up to 120 hpf is accompanied by the appearance of epithelial cells and therefore of the xenobiotic-metabolizing machinery (i.e., CYP3A65, zfPXR and zfAhR2) (Wallace et al. 2005), confirming the trends observed in the literature with whole mount *in situ* hybridization of *cyp3a65*, as well as RT-qPCR protocols (Tseng et al. 2005; Glisic et al. 2016).

As an *in vivo* zebrafish assay, EMERGE offers the advantage of assessing the intestinal effects of chemicals following whole-body exposure, thereby providing a greater consideration of metabolic processes compared to *in vitro* cell-based assays. Furthermore, while PXR exhibits the highest degree of interspecies difference among nuclear hormone receptors (Ekins et al. 2008), zebrafish shows greater proximity in amino acid identity to human PXR than many other non-mammalian models such as *Xenopus laevis* (Moore et al. 2002) or other
<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Representative concentration-responses obtained in EMERGE for biocides/pesticides. GFP expression in zebrafish embryos exposed to (A) chlorpyrifos (CPF); (B) tributyltin (TBT); (C) triclosan (TCS). \* denotes significant differences as compared to control DMSO 0.01%; Kruskal Wallis test followed by Dunn's test; $P \le 0.05$ (*), $P \le 0.01$ (**), $P \le 0.001$ (***), and $P \le 0.0001$ (****). Data are represented as violon plot with each point representing fluorescent intensity in individual embryo, expressed as fold GFP intensity above control; solid lines represent the median of each test condition. Outliers (detected with GraphPad Prism: ROUT method with $Q = 1\%$) were removed. DDE is not represented as results varied across experiments. All independent experiments can be found in supplementary material.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 11.** Concentration-responses obtained in EMERGE for pharmaceuticals. GFP expression in zebrafish embryos exposed to (A) amiodarone hydrochloride (AMD); (B) clotrimazole (CLO); (C) clobetasol propionate (CLOBE); (D) fluticasone propionate (FLU). \* denotes significant differences as compared to control DMSO 0.01%; Kruskal Wallis test followed by Dunn's test; $P \le 0.05$ (*), $P \le 0.01$ (**), $P \le 0.001$ (***), and $P \le 0.0001$ (****). Data are represented as violon plot with each point representing fluorescent intensity in individual embryo, expressed as fold GFP intensity above control; solid lines represent the median of each test condition. Outliers (detected with GraphPad Prism: ROUT method with $Q = 1\%$) were removed. DGT is not represented as results varied across experiments. All independent experiments can be found in supplementary material.</figcaption></figure>

fish such as *Oryzias latipes* (Lille-Langøy et al. 2019), not to mention certain teleost species in which PXR is completely absent (e.g., *Gasterosteus aculeatus*) (Eide et al. 2018).

Each model, including the EMERGE assay, displays potential limitations. While interspecies differences between the various PXR

receptors may represent a potential limitation in the EMERGE assay ability to predict substance effects in humans, it should be noted that, although mammalian models are structurally closer to hPXR, there is no ideal animal model for this receptor (Moore et al. 2002). Secondly, the protein expression of *cyp3a65* has not been investigated due to the lack
<figure><img src="image_14.png" /><figcaption>**Fig. 12.** Representative concentration-responses obtained in EMERGE for metals (A), phthalates (B) and perfluorinated compounds (C). GFP expression in zebrafish embryos exposed to (A) cadmium chloride (CdCl2); (B) di(2-ethylhexyl)phthalate (DEHP); (C) perfluorooctanoic acid (PFOA). \* denotes significant differences as compared to control DMSO 0.01 %; Kruskal Wallis test followed by Dunn's test; $P \le 0.05$ (\*), $P \le 0.01$ (\*\*), $P \le 0.001$ (\*\*\*\*) and $P \le 0.0001$ (\*\*\*\*\*). Data are represented as violon plot with each point representing fluorescent intensity in individual embryo, expressed as fold GFP intensity above control; solid lines represent the median of each test condition. Outliers (detected with GraphPad Prism: ROUT method with $Q = 1$ %) were removed. All independent experiments can be found in supplementary material.</figcaption></figure>

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

of specific antibodies; further studies using immunohistochemistry staining would enable to identify the type of intestinal cells expressing specifically CYP3A65. In addition, *cyp3a65* expression has not been investigated after 120 hpf, which corresponds to post-embryonic stages, and raises the question of the toxicological relevance of extending the chemical exposure phase beyond 120 hpf in the EMERGE assay. *Nawaji et al.* (2020) reported a high *cyp3a65* expression at embryonic stages and a decrease in zebrafish adult liver. Moreover, *Verbueken et al.* (2018) indicated a *cyp3a65* peak of expression around 120 hpf and also noted that *cyp3a65* whole-body expression was relatively stable over the first 30 days of development, despite the growth burst, suggesting significant tissue-specific activity. Further work is needed to investigate *cyp3a65* expression in the intestine at juvenile and adult stages, but these studies suggest an important role of this enzyme at embryonic stage.

Most importantly, EMERGE was designed to detect early receptor-mediated transcriptional responses after exposure to test chemicals and not to predict adverse outcomes or chronic metabolic disorders. EMERGE therefore contributes to a better understanding of the MoAs of potential MDCs but must be complemented by other assays to cover later events and metabolic outcomes. For that, the consequences of a *cyp3a65* dysregulation should be studied in more detail from a risk assessment perspective, by investigating intestinal outcomes (e.g., enzymatic activity, gut inflammation, nutrient transport, cholesterol metabolism, gut microbiome dysbiosis).

## 5.2. Biological activity of tested chemicals in EMERGE

Besides these limitations, our study demonstrated the usefulness of the EMERGE assay to screen and identify chemicals for their potential metabolic activity in the developing intestine. Among the 22 test substances, 18 were able to modulate *cyp3a65* expression either by inducing (14) or by inhibiting (4) GFP expression. To the best of our knowledge, for most of the substances tested, these results represent the first report in the literature on the modulation of *cyp3a65* expression, highlighting the significance of the EMERGE assay.

With similarities to *cyp3a4* gene in humans, *cyp3a65* regulation involves multiple transcription factors, including ligand-regulated

receptors such as zfPXR, zfAhR2, and zfGR (Erradhouani et al. 2025). Our results are consistent with this multi-pathway regulation, as shown by:

* The potent induction of GFP by zfAhR agonists (TCDD, PeCDD, BaAQ and 5,12-NQ);

* The moderate induction by zfPXR agonists (CLO and DGT);

* The strong inhibition by zfGR agonists (FLU and CLOBE).

## 5.3. Active substances in EMERGE

### 5.3.1. Dioxins and PAHs

Among active substances in EMERGE, dioxins are known to disrupt bile acid metabolism and glucose transporter sodium-glucose cotransporter 1 in the intestine increasing glucose uptake and hepatic bile acid levels, as shown in C57BL/6 mice (Fader et al. 2017; Ishida et al. 2005). PAHs can promote intestinal inflammation and dysbiosis as shown in the same mouse model (Ribière et al. 2016), as well as alteration of intestinal enzymatic lipid digestion, as observed in rainbow trout (De Gelder et al. 2017). In EMERGE, these substances induce intestinal *cyp3a65* expression at pM to nM concentrations (Fig. 7 and Table 1), showing that the intestine is a target of these chemicals. EMERGE therefore provides a relevant model to study intestinal responses to MDCs. Moreover, both dioxins and PAHs are AhR ligands (Mandal 2005; Piterse et al. 2013), which not only confirm the regulation of *cyp3a65* by zfAhR2 in zebrafish, but also reinforce the idea that metabolic disorders can occur through AhR activation (Gao et al. 2023).

### 5.3.2. Pharmaceuticals

Clotrimazole (CLO) has been shown to modify the expression of glucose transporter 2 in Caco-2 human intestinal cells (Gonçalves et al. 2012) and to increase P-glycoprotein isoform in rainbow trout intestine (Love et al. 2021). In addition, its ability to inhibit CYP3A activity makes this substance a potent MDC, capable of disrupting numerous metabolic processes, as shown in rainbow trout and rodent hepatic microsomes (Turan et al. 2001; Burkina et al. 2013). Few studies have examined the effects of desogestrel (DGT) on intestinal metabolism. However, in
humans, the contraceptive is known to decrease insulin sensitivity (Cagnacci et al. 2009). In EMERGE, exposure to CLO and DGT leads to increased *cyp3a65* expression, likely due to their ability to bind and activate zfPXR in *in vitro* reporter gene assays (Creusot et al. 2021), thereby providing new information about their MoAs at the intestinal level.

On the other hand, exposure to synthetic glucocorticoids (FLU and CLOBE) strongly inhibits *cyp3a65* constitutive expression in EMERGE. This further supports our recent data showing thatzfGR is required in the inhibition of *cyp3a65* by fluticasone propionate and hypothesized that the mechanism through whichzfGR regulates the expression of *cyp3a65* implies HNF4α, a transcription factor known to be involved with GR in the regulation of *cyp3a4* in humans (Erradhouani et al., 2025). Interestingly, glucocorticoids are well-known to disrupt multiple aspects of metabolic physiology through glucocorticoid receptor activation (La Merrill et al. 2025), notably insulin signaling, control of appetite or adipose and intestinal physiology. Our results highlight the importance of investigating the metabolic effects of GR-ligand chemicals in the intestine.

Among pharmaceuticals, amiodarone (AMD) is well-known to induce liver steatosis as shown in human and mice models (Anthérieu et al. 2011; Vitins et al. 2014). A recent study indicates that AMD-induced steatohepatitis is associated with intestinal alterations (e.g., disruption of microbiome and intestinal permeability) (Kawauchi et al. 2025), suggesting that the intestine contributes to the pathogenesis of steatohepatitis. Interestingly, AMD is metabolized by CYP3A4, human orthologue of CYP3A65, by N-deethylation leading to toxic metabolites involved in its hepatotoxicity (Wu et al. 2016; Zahno et al. 2011). The induction of *cyp3a65* observed in EMERGE suggests an involvement of AMD metabolism by CYP3A65 in the intestine. To our knowledge, this extra-hepatic metabolization of AMD *via* CYP3A4 has not been reported to date in humans. In any case, the precise mechanism by which AMD induces the expression of zebrafish intestinal *cyp3a65* is not known but could involve the PXR pathway, AMD being able to transactivate hPXR in cell-based reporter gene assay at µM concentrations (ToxCast TOX21 assay: SM Table S3).

### 5.3.3. Bisphenols

Exposure to bisphenols has been identified to cause alteration of intestinal barrier structure therefore causing intestinal inflammation and modification of its absorption function as shown in various models such as Sprague-Dawley rats or human colon NCM460 cells (Kamaludin et al. 2022; Liu et al. 2022).

Among the six bisphenols tested, four could induce the intestinal *cyp3a65* expression in EMERGE (i.e., BPA, BPB, BPC, BPF), while BPS and TCBPA were totally inactive. Bisphenols are well-known for their estrogenic activity in zebrafish embryos (Le Fol et al. 2017; Pinto et al. 2019) and the metabolic effects of BPA in zebrafish embryos have been reported notably through its ability to disrupt lipid metabolism, yolk lipid absorption, metabolic signaling and food intake (Boom et al. 2024; Martinez et al. 2020; Ortiz-Villanueva et al. 2017). Moreover, the study of Mu et al. (2022) noted some specific intestinal effects in zebrafish (e. g., inflammatory responses and activation of lipid uptake and absorption pathways) after exposure to some BPA analogues. In EMERGE, their ability to induce *cyp3a65* in the intestine could reflect their interaction with the ligand-activated transcriptional factors involved in the regulation of *cyp3a65*. Several studies have reported activation of the AhR signaling pathway *in vitro* by several bisphenols in HepG2 and transfected rat hepatoma cells (Rosenmai et al. 2014; Yu et al., 2021a), an effect which is however not reported in other hAhR transactivation assays (SM Table S3, Balaguer et al., unpublished data). Recently, a large-scale screening study suggests that AhR activation occurs after exposure to a few numbers of bisphenols, and only at high concentrations with low specificity (Srebny et al. 2025), suggesting a limited toxicological relevance of this pathway. In contrast, several bisphenols have been shown to activate hPXR using different *in vitro* reporter transactivation

assays (Grimaldi et al. 2019; Kojima et al. 2019; SM Table S3), but not mouse PXR (Sui et al. 2012). It is therefore likely that interaction of bisphenols with PXR could explain their activity in EMERGE. However, it would be advisable to test their activity through zfPXR to support this hypothesis as significant species-specific differences exist between the ligand binding domain of PXR in zebrafish and humans (Bainy et al. 2013, Creusot et al., 2021). In any case, the study from Mu et al. (2022) and the data reported herein using EMERGE showed that beyond their well-characterized estrogenic activity, some bisphenols elicit metabolic activity in the intestine of zebrafish (e.g., activation of lipid uptake and absorption pathways, inflammatory responses and *cyp3a65* induction). This is in line with the recent study by Srebny et al. (2025) showing that, besides estrogenicity, some BPA alternatives could have additional modes of action (e.g., PPARγ agonism, mitochondrial effects), highlighting that bisphenols should not just be considered as estrogenic endocrine disruptors.

### 5.3.4. Biocides/Pesticides and other substances

Chlorpyrifos (CPF) appears to promote dysbiosis by altering the intestinal barrier integrity, leading to insulin resistance and increased body weight in C57Bl/J6 mice (Liang et al. 2019). Interestingly, CPF increases *cyp3a65* expression in EMERGE and has been shown to induce the expression of *pxr* and PXR-regulated *cyp2k1* in rainbow trout intestine (De Anna et al. 2021). In the same way, PFOA is responsible for various metabolic effects in humans, including impaired glucose metabolism and lipid oxidation (Chen et al. 2020), while in CD-1 mice, it has been shown that developmental exposure to PFOA increased serum leptin and insulin (Hines et al. 2009). In EMERGE, PFOA increases *cyp3a65* expression, reflecting the upregulation of hepatic *pxr* and *cyp3a* genes observed in exposed juvenile salmon (Mortensen et al. 2011). Thus, results for both CPF and PFOA support the idea that the PXR signaling pathway plays a central role in mediating their effects on *cyp3a65* expression. Nonetheless, Mortensen and Arukwe (2006) found an up-regulation of these genes (i.e., *pxr* and *cyp3a*) in juvenile salmon liver exposed to 4,4'-DDE while in EMERGE this compound was found to inhibit *cyp3a65* expression in the intestine.

Triclosan (TCS) has been identified in hepatotoxicity, by inducing steatosis, hepatic inflammation and disruption of lipid metabolism in zebrafish (Ho et al. 2016; Sun et al. 2020). In mice, TCS was also shown to exacerbate high-fat diet induced nonalcoholic fatty liver disease (Yueh et al. 2020). Moreover, a recent study reported that TCS exposure can induce lipid accumulation not only in the liver but also in the zebrafish intestine (Li et al. 2024). In EMERGE, TCS modulates *cyp3a65* expression in the intestine, as shown by GFP induction. Several studies showed that TCS activate hPXR (Creusot et al. 2010; Jacobs et al. 2005; Paul et al. 2013), which could explain its effect in EMERGE, although its activity on zfPXR still needs investigation.

CdCl₂ is known to promote hyperlipidaemia and fatty liver as shown in zebrafish (Kim et al. 2018), while in the intestine, various rodent models and human cell models show modified permeability and dysbiosis leading to intestinal inflammation and disturbance of energy metabolism (Tinkov et al. 2018). In EMERGE, CdCl₂ was active by inhibiting GFP in the intestine. CdCl₂ has been shown to disrupt the hypothalamic-pituitary-interrenal (HPI) axis in fish (Liu et al. 2016; Sandhu et al. 2014) and interact with the hGR in TOX21 GR BLA antagonist ratio assay (SM Table S3), but was found inactive on hGR in another study (Zhang et al. 2018). Currently, it is difficult to attribute a specific MoA to CdCl₂ and explain its inhibitory effect on *cyp3a65* expression.

### 5.4. Inactive substances in EMERGE

Among the compounds tested, BPS, TCBPA, DEHP and TBT were consistently inactive in EMERGE. The inactivity of BPS and TCBPA shows that differences can occur even within structurally related chemical families, suggesting that *cyp3a65* modulation cannot be
predicted only from chemical structure. Concerning BPS, it has been reported to have a different toxicokinetic profile in zebrafish embryos compared to BPA, BPAF and BPF (Billat et al. 2023), which could explain the differences in bioactivity observed on *cyp3a65* in EMERGE. Consistently, BPS was also found to be less estrogenic than its congeners in several zebrafish embryo studies (Le Fol et al. 2017; Moreman et al. 2017).

Di-(2-ethylhexyl) phthalate (DEHP) and tributyltin (TBT) are two well-established metabolic disrupting chemicals (MDCs). The metabolite of DEHP, MEHP, and TBT were primarily well documented for their obesogenic effects on adipocytes through high affinity for hPPARγ (Feige et al. 2007; Grün and Blumberg 2006; Kanayama et al. 2005), while TBT also exhibited a strong affinity for RXR, the heterodimeric partner of PPARγ and PXR in humans (Delfosse et al. 2021). Moreover, MEHP can also activate zebrafish PPARγ (Garoche et al. 2021). Beyond adipogenic effects, both DEHP and TBT have also been linked to intestinal alterations. In mice, DEHP exposure induces dysbiosis and gut barrier disruption, leading to intestinal inflammation (Cheng et al. 2024). Similar alterations were reported in rats, with DEHP-induced dysbiosis was associated with bile acid metabolism disruption and cholesterol imbalance (Yu et al. 2021b). For TBT, exposure also induced gut microbiome dysbiosis in mice in several studies, in link with increased body weight, dyslipidemia, and impaired glucose and insulin homeostasis (Guo et al. 2018; Zhan et al. 2020).

In EMERGE, both DEHP and TBT were inactive on *cyp3a65* expression (Figs. 12B and 10B). This inactivity suggests that intestinal *cyp3a65* is not a relevant target of these compounds in the experimental conditions of EMERGE assay (i.e., short-term exposure at early developmental stages), which may represent a limit of the assay in detecting metabolic effects of these compounds. Moreover, the absence of activity of DEHP and TBT in EMERGE suggests that PPARγ and RXR are not key regulators of *cyp3a65* expression in zebrafish intestine as in humans for *cyp3a4* (Chen et al. 2014).

### 5.5. Comparison of the biological activity of chemicals on zebrafish *cyp3a65* (EMERGE assay) and human *cyp3a4*

An important question that arises from this study is the relevance of the data obtained using EMERGE with regard to a possible effect on this gene in humans. In this perspective, we compared our results with available data from human-based assays targeting *cyp3a4* (Table 2). Among the 22 tested substances, information on their effect on *cyp3a4* expression in humans was available for only 12, mostly from *in vitro* liver models. This highlights the limited information on this gene at the intestinal level in humans. Nevertheless, we found that among the 12 substances tested in EMERGE and human cell-based assays, 8 were active on both *cyp3a65* and *cyp3a4*, with 7 being capable of inducing both *cyp3a65* and *cyp3a4* (Table 2). For the other 4 substances (BPF, DEHP, 4,4'-DDE and TBT), the differences are noteworthy and could be explained by:

* Interspecies differences in receptors interaction to chemicals, notably PXR, which shows significant differences between the ligand binding domain of zebrafish and humans (Bainy et al. 2013; Creusot et al. 2021);

* Tissue/cell-specific differences between the intestine and liver. Those types of differences have already been reported between liver (HepG2) and intestinal (LS180) human cells for PXR response to some substances (Sui et al. 2015);

* Differences between *in vitro* and *in vivo* contexts, which could be for example the case for TBT, active on *cyp3a4* expression in intestinal LS147T cells but not on *cyp3a65* expression *in vivo*, although it has been shown to induce *cyp3a65* *in vitro* (Marqueño et al. 2021);

* Differences in regulation of CYP3A4 in humans and CYP3A65 in zebrafish. Notable differences exist, particularly in the interaction between zfPXR and zfAhR2 receptors, which interact in a complex manner to regulate *cyp3a65* expression in zebrafish embryos (Erradhouani et al. 2025; Kubota et al. 2015).

<caption>**Table 2**</caption>

Comparison of the effects (median maximal fold-change) of chemicals on zebrafish *cyp3a65* gene expression based on the EMERGE assay and human *cyp3a4* gene expression based on the literature.

<table><thead><tr><th rowspan="2">Family</th><th rowspan="2">Chemicals</th><th colspan="2">cyp3a65 (EMERGE)</th><th colspan="2">cyp3a4 (literature)</th><th rowspan="2">In vitro human models</th><th rowspan="2">Methods</th><th rowspan="2">References</th></tr><tr><th>Induction</th><th>Inhibition</th><th>Induction</th><th>Inhibition</th></tr></thead><tbody><tr><td>Dioxins</td><td>TCDD</td><td>2.3</td><td></td><td>3</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td>(Kumagai et al., 2012)</td></tr><tr><td rowspan="3">Oxy-PAHs</td><td>PeCDD</td><td>6.3</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>5,12-NQ</td><td>3.0</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>BaAQ</td><td>4.0</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Bisphenols</td><td>BPA</td><td>1.9</td><td></td><td>4.8</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td>(Yu et al. 2021a)</td></tr><tr><td>BPF</td><td>4.2</td><td></td><td>4.5</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td></td></tr><tr><td>BPS</td><td>no effect</td><td></td><td>2.8</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td></td></tr><tr><td>BPB</td><td>3.3</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>BPC</td><td>3.0</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>TCBPA</td><td>no effect</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Biocides</td><td>CPF</td><td>3.3</td><td></td><td>5.5</td><td></td><td>HepG2 (liver)</td><td>Branched DNA assay</td><td>(Das et al., 2008)</td></tr><tr><td>DDE</td><td></td><td>0.075</td><td>no effect</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td>(Shabbir et al., 2005)</td></tr><tr><td>TBT</td><td>no effect</td><td></td><td>weak induction</td><td></td><td>LS174T (intestine)</td><td>RT-PCR</td><td>(Delfosse et al., 2021)</td></tr><tr><td rowspan="5">Pharma</td><td>TCS</td><td>4.3</td><td></td><td>4.36</td><td></td><td>MHCC97H (liver)</td><td>qPCR</td><td>(Wu et al., 2018)</td></tr><tr><td>AMD</td><td>3.4</td><td></td><td>no value</td><td></td><td>HepG2 (liver)</td><td>RT-PCR</td><td>(Zahno et al. 2011)</td></tr><tr><td>CLO</td><td>5.15</td><td></td><td>18</td><td></td><td>LS174T (intestine)</td><td>RT-PCR</td><td>(Svecova et al., 2008)</td></tr><tr><td>DGT</td><td>no effect</td><td></td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>CLOBE</td><td></td><td>0.047</td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Metal Phthalates</td><td>FLU</td><td></td><td>0.06</td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>CdCl<sub>2</sub></td><td></td><td>0.15</td><td>no information</td><td></td><td></td><td></td><td></td></tr><tr><td>Perfluorinated compounds</td><td>PFOA</td><td>2</td><td></td><td>13</td><td></td><td>HepG2 (liver)</td><td>Reporter gene assay (LUC)</td><td>(Cooper et al. 2008)</td></tr><tr><td></td><td></td><td></td><td></td><td>0.2</td><td></td><td>HepaRG (liver)</td><td>qPCR</td><td>(Franco et al., 2020)</td></tr></tbody></table>
These observations suggest that the EMERGE assay could provide valuable insights into the effects of substances on *cyp3a4* in humans, particularly at the intestinal level, which remains under-explored for this CYP. As several substances tested were able to modulate *cyp3a65* in the zebrafish intestine, it suggests that they could also impact intestinal metabolism in humans. Interestingly, CYP3A4 has been recognized as a major enzyme driving intestinal metabolism in humans, and a recent study has highlighted the need to better integrate intestinal toxicokinetics into *in vitro* to *in vivo* extrapolation models (Korol-Bexell et al. 2025). In this context, EMERGE could provide important information regarding the contribution of extra-hepatic metabolism, which influences the overall toxicokinetics of environmental contaminants.

### 5.6. Scientific and regulatory perspectives for EMERGE assay

Based on the transgenic *tg(cyp3a65:GFP)* line, the EMERGE assay has the potential to enhance our understanding of *cyp3a65* expression and regulation but also to provide a valuable new tool for advancing knowledge in the metabolic toxicology of the zebrafish model. In humans, CYP3A4 is involved in the metabolism of both xenobiotics and endogenous compounds such as bile acids, and these processes can be disrupted by MDCs (Chen et al. 2014). The changes in *cyp3a65* expression observed in response to the xenobiotics screened in EMERGE strongly suggest that CYP3A65 plays a significant role in xenobiotic metabolism. However, as previously mentioned, its involvement in intestinal metabolism and the potential disruption caused by these substances still require further investigation. Future experiments in adult zebrafish will help establish a potential link between CYP3A65 and other endogenous substances in the intestine (e.g., cholesterol and bile acids).

As the first established vertebrate-based assay for evaluating metabolic disruptors in the intestine, EMERGE addresses a critical gap in current regulatory testing strategies, which primarily focus on hepatic or adipose tissue endpoints. Its ability to integrate multiple metabolic pathways and detect activation and inhibition of constitutive *cyp3a65* expression makes it particularly valuable for mechanistic hazard characterization in a more physiological view. In line with initiatives of the OBERON European project and the current needs to develop new approach methodologies (NAMs), EMERGE offers a novel tool to assess chemical effects beyond the classical EATS (estrogen, androgen, thyroid, steroidogenesis) pathways in endocrine disruption.

Importantly, its application could extend beyond individual chemicals, offering a relevant approach for assessing mixture effects in an *in vivo* context. By providing tissue-specific information and capturing toxicodynamic interactions (e.g., interaction between transcription factors), EMERGE could contribute to a more comprehensive evaluation of chemical interactions in hazard assessment.

From a regulatory perspective, EMERGE is not currently intended for stand-alone regulatory validation. Instead, it is designed as a screening tool to be used in combination with other *in vitro* and *in vivo* approaches, particularly in tiered testing strategies, in line with Integrated Approaches to Testing and Assessment (IATA). The ReadEDTest self-assessment questionnaire (Crouzet et al. 2023) developed within the OBERON project confirmed several strengths of the assay (e.g., fish line and monitoring, test method controls), while identifying areas needing improvement, including standard operating procedures (SOPs), data management and inter-laboratory reproducibility (SM Fig. S23). Further work is also required to validate this assay as a potential new assay to identify substances that affect the intestinal metabolism.

## 6. Conclusion

In this study, we successfully developed a stable *tg(cyp3a65:GFP)* transgenic zebrafish line that expresses GFP under the control of *cyp3a65*. Our results show an intestinal expression of GFP from 72 to 120 hpf which faithfully reflects the dynamic expression of the

endogenous gene during embryo development. Using fluorescence microscopy and RT-qPCR, we demonstrated that clotrimazole (zfPXR agonist) and TCDD (zfAhR2 agonist) were able to induce *cyp3a65* expression, confirming the regulating role of these signaling pathways. On this basis, we further developed a zebrafish embryo-based assay, called EMERGE, to assess the response of *cyp3a65* after 48 h of exposure to 22 MDCs. The screening revealed that a wide range of chemicals modulates *cyp3a65* expression in the intestine. Given the current lack of *in vivo* assays to screen the impact of chemicals on intestinal metabolism, EMERGE could serve as a novel tool in MDCs hazard assessment as it provides new information regarding the biological activity of chemicals in the intestine of an embryonic vertebrate model. Such assay could be relevant into an integrated testing strategy combining *in silico*, *in vitro*, and *in vivo* assays. However, additional studies should investigate more chemicals and the role of CYP3A65 in long-term metabolic outcomes.

As the impact of MDCs on human CYP3A4 in the intestine has not been well studied, future research should complete our findings by testing MDCs in mammalian intestinal models to confirm the relevance of the EMERGE assay for human health assessment.

### CRediT authorship contribution statement

**Chedi Erradhouani:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Florian Geffroy:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Benjamin Piccini:** Methodology, Investigation. **Nathalie Hinfray:** Methodology, Investigation. **Edith Chadili:** Methodology, Investigation. **Patrick Balaguer:** Writing – review & editing, Funding acquisition. **Frédéric Sohm:** Writing – review & editing, Resources, Methodology, Investigation. **Selim Aït-Aïssa:** Writing – review & editing, Project administration, Funding acquisition. **Xavier Coumoul:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **François Brion:** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, 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.

### Acknowledgment

This study was funded by the H2020 OBERON project (grant agreement no 825712) and the European Partnership for the Assessment of Risks from Chemicals (PARC) and has received funding from the European Union's Horizon Europe research and innovation program under Grant Agreement No 101057014. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The transgenic model was first developed within the project PestR from French National Research Program for Endocrine Disruptors (PNRPE). C.E. and F.G. received a doctoral fellowship from INERIS (P190 – Ecotoxicologie). The authors acknowledge INERIS, INSERM and Université Paris-Cité for their financial and scientific support in this project. The authors would like to thank the reviewers for their constructive comments on the manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2025.109872.
# Data availability

Data will be made available on request.

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