# Extracellular Vesicles as a potential source of biomarkers for endocrine disruptors in MASLD: a short review on the case of DEHP

Pierre-Etienne Merret, Lydie Sparfel, Catherine Lavau, Dominique Lagadic-Gossmann, Corinne Martin-Chouly

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Pierre-Etienne Merret, Lydie Sparfel, Catherine Lavau, Dominique Lagadic-Gossmann, Corinne Martin-Chouly. Extracellular Vesicles as a potential source of biomarkers for endocrine disruptors in MASLD: a short review on the case of DEHP. Biochimie, 2024, 10.1016/j.biochi.2024.09.009 hal-04718117

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Submitted on 23 Jan 2026

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<figure><img src="image_2.png" /></figure>
# Extracellular vesicles as a potential source of biomarkers for endocrine disruptors in MASLD: A short review on the case of DEHP

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

Pierre-Etienne Merret, Lydie Sparfel, Catherine Lavau, Dominique Lagadic-Gossmann **, Corinne Martin-Chouly *

Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail) - UMR_S 1085, F-35000, Rennes, France

## ARTICLE INFO

**Article history:**

Received 14 February 2024

Received in revised form

28 August 2024

Accepted 20 September 2024

Available online 21 September 2024

Handling Editor: C. Forest

**Keywords:**

MASLD

Extracellular vesicles

Biomarkers

DEHP

Endocrine disruptors

## ABSTRACT

Metabolic dysfunction—Associated Steatotic Liver Disease (MASLD) is a chronic disease with increasing prevalence and for which non-invasive biomarkers are needed. Environmental endocrine disruptors (EDs) are known to be involved in the onset and progression of MASLD and assays to monitor their impact on the liver are being developed. Extracellular vesicles (EVs) mediate cell communication and their content reflects the pathophysiological state of the cells from which they are released. They can thus serve as biomarkers of the pathological state of the liver and of exposure to EDs. In this review, we present the relationships between DEHP (Di(2-ethylhexyl) phthalate) and MASLD and highlight the potential of EVs as biomarkers of DEHP exposure and the resulting progression of MASLD.

© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Contents

<table><tr><td>1. Introduction</td><td>128</td></tr><tr><td>2. DEHP is an endocrine disruptor targeting the liver</td><td>128</td></tr><tr><td>2.1. Hepatotoxicity of DEHP and MEHP</td><td>129</td></tr><tr><td>2.2. DEHP is a metabolic disruptor involved in the development of MASLD</td><td>129</td></tr><tr><td>3. Extracellular vesicles and MASLD</td><td>129</td></tr><tr><td>4. EV-derived miRNAs as putative biomarkers of DEHP exposure in MASLD</td><td>130</td></tr><tr><td>4.1. Circulating or EV-derived miRNAs in MASLD</td><td>130</td></tr><tr><td>4.2. EV-derived miRNAs in the context of DEHP exposure</td><td>131</td></tr><tr><td>5. EV proteins as putative biomarkers of DEHP exposure in MASLD</td><td>132</td></tr><tr><td>5.1. EV proteins in MASLD</td><td>132</td></tr><tr><td>5.2. EV proteins associated with DEHP exposure</td><td>133</td></tr><tr><td>6. EV lipids as putative biomarkers of DEHP exposure in MASLD</td><td>133</td></tr><tr><td>6.1. EV lipids in MASLD</td><td>133</td></tr><tr><td>6.2. Effect of DEHP on lipids</td><td>133</td></tr><tr><td>7. Use of those candidates as biomarkers</td><td>134</td></tr><tr><td>8. Clinical applications in liver diseases</td><td>134</td></tr><tr><td>9. Conclusion</td><td>134</td></tr><tr><td>Funding</td><td>135</td></tr><tr><td>CRediT authorship contribution statement</td><td>135</td></tr><tr><td>Declaration of competing interest</td><td>135</td></tr></table>

<footnote>* Corresponding author. UMR_S 1085, 2 avenue du Pr Léon Bernard, 35043, Rennes, France.</footnote>

<footnote>** Corresponding author. UMR_S 1085, 2 avenue du Pr Léon Bernard, 35043, Rennes, France. E-mail addresses: dominique.lagadic@univ-rennes.fr (D. Lagadic-Gossmann), corinne.chouly@univ-rennes.fr (C. Martin-Chouly).</footnote>
<table><tr><td>Acknowledgments</td><td>135</td></tr><tr><td>References</td><td>135</td></tr></table>

## Abbreviations

<table><tr><td>DEHP</td><td>Di (2-ethylhexyl) phthalate</td></tr><tr><td>ED</td><td>Endocrine Disruptor</td></tr><tr><td>EV</td><td>Extracellular Vesicle</td></tr><tr><td>MASH</td><td>Metabolic dysfunction-Associated SteatoHepatitis</td></tr><tr><td>MASLD</td><td>Metabolic dysfunction-Associated Steatotic Liver Disease</td></tr><tr><td>MEHP</td><td>Mono (2-ethylhexyl) phthalate</td></tr><tr><td>miRNAs:</td><td>microRNAs</td></tr><tr><td>PVC</td><td>Polyvinyl chloride</td></tr><tr><td>S1P:</td><td>Sphingosine 1-Phosphate</td></tr><tr><td>SK1:</td><td>Sphingosine Kinase 1</td></tr><tr><td>TF:</td><td>Tissue Factor</td></tr></table>

# 1. Introduction

Concern over metabolic diseases has steadily risen during the past decades as growing numbers of individuals suffer from the likes of diabetes, obesity or non-alcoholic fatty liver disease (NAFLD). In 2023, multinational liver associations proposed that metabolic dysfunction-associated steatotic liver disease (MASLD) should replace the term NAFLD; the name chosen to replace non-alcoholic steatohepatitis was metabolic dysfunction-associated steatohepatitis (MASH) [1]. Such a change in terminology was based on the will to take into account cardio-metabolic disorder criteria rather than only focusing on alcohol consumption. With a global prevalence estimated at 30% of the population [2], MASLD is now considered an epidemic whose impact on public health is of great concern.

As common as it might be, MASLD is by no means a simple pathology to diagnose. In fact, MASLD encompasses a complex array of chronic liver diseases, not tied to genetics or the consumption of drugs or alcohol, that range from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH). The complexity of this array stems from the apparent continuity in the appearance of histopathological elements accompanying the evolution of MASLD, starting from an accumulation of lipid droplets (steatosis) and evolving towards inflammation, cell death and the buildup of fibrotic tissue (MASH) [3–5].

With poor consensus on grades and stages that can be used as references during diagnosis, and with biopsy being the only current reliable method to explore liver status, less invasive and more precise diagnostic tools are needed to care for patients suffering, or at risk of suffering, from MASLD.

Regarding the risk factors leading to the development of MASLD, a growing amount of evidence links exposure to environmental toxicants, in particular endocrine disrupters (EDs), to metabolic disorders such as MASLD [6,7]. Epidemiological studies notably link the onset of MASLD to prenatal exposure to phthalates [8]. This mini review will focus on the Di (2-ethylhexyl) phthalate (DEHP) compound, a well-known endocrine disruptor, and explore the potential for biomarkers associated with extracellular vesicles (EVs) to help diagnose and monitor MASLD related to EDs. Key elements

will be briefly introduced followed by an overview of candidate biomarkers and their possible clinical applications.

# 2. DEHP is an endocrine disruptor targeting the liver

DEHP is a commonly used plasticizer [9] with a wide array of applications, ranging from the production of everyday polyvinyl chloride (PVC) items to that of medical equipment [9,10]. As a plasticizer, DEHP is not covalently bound to the polymer matrix, and can thus leach out in the environment over time [11]. Most notably, DEHP can transfer to materials passing through tubing or held within containers, thus causing patients or consumers to be exposed over potentially long periods of time [12]. As shown by its vapor pressure and water solubility, DEHP is very slightly volatile and slightly soluble in water but its partition coefficient n-octanol/water of 7.5 indicates its strong affinity for organic matter and lipids; it is thus found in various biological fluids (ECHA, 2014). Several European texts now regulate the use of some phthalates used as plasticizers, in everyday consumer goods and in health products. That is the case of DEHP that is regulated under REACH (registration, evaluation, authorisation and restriction of chemicals, Regulation (EC) No 1907/2006) after classification as a substance of very high concern (SVHC); so, since 2015 authorization is required for the use of DEHP and other EDs (REACH Annex XIV). The latest amendment of REACH Annex XVII took effect in July 2020, restricting the general use of DEHP in consumer products [13]. However, DEHP is still used in many countries all around the world.

Exposure of the general population to DEHP is ubiquitous, with the oral route being the main route of exposure through the consumption of food and water [14]. Special cases nevertheless exist, such as patients undergoing frequent dialysis or chronic transfusion therapy [12] and professional exposure of workers in certain industries. In those latter cases, other routes can be involved such as the circulatory, dermal or pulmonary ones.

As a lipophilic compound, DEHP is notably metabolized in the gastro-intestinal tract by pancreatic lipases into mono(2-ethylhexyl)phthalate (MEHP) [15] and 2-ethyl-hexanol, with the former compound being of interest from a toxicological standpoint [16,17]. Distribution of DEHP and its metabolites occurs through the bloodstream and reaches multiple organs, with documented toxicity towards the testes, ovaries, kidneys, brain, heart, and liver [17]. Most studies on DEHP pertain to its potential as an endocrine disruptor; that is, a molecule able to impact the functions of the endocrine system, often leading to adverse effects. According to the world health organization (WHO) "An endocrine disruptor is an exogenous substance or mixture which alters functions of the endocrine system and consequently induces harmful effects on the health of an intact organism (or) of its descendants" [18]. In 2018, the European Commission adopted in 2018 a new definition for the determination of endocrine-disrupting properties, aligned with the WHO definition and repealing therefore an "interim" regulatory criteria. Thus, if a phytopharmaceutical active substance is identified as having an endocrine-disrupting effect, commercialization is not authorized in the European Union [19]. DEHP has thus been associated with numerous health disorders, notably diabetes, metabolic syndrome and obesity [7]. Beyond an impact on energy metabolism that can contribute to obesity, DEHP has also been found to negatively affect reproductive functions, with documented
links with the anogenital distance, endometriosis and low testosterone levels [20–23]. Regarding the liver, DEHP and its main metabolite, MEHP, have been related to hepatotoxicity as well as MASLD [24,25].

## 2.1. Hepatotoxicity of DEHP and MEHP

Human studies on the hepatotoxicity of DEHP have mostly investigated the impact of high doses of DEHP in the circulatory system due to medical procedures involving DEHP-containing plastics such as extracorporeal membrane oxygenation, parenteral nutrition or neonatal intensive care. Those scenarios show associations between exposure to DEHP and the development of cholestasis or the incidence and severity of hepatoblastoma [26]. Prenatal exposure to the main metabolite MEHP in humans was also linked to adverse metabolic profiles including the development of an unfavourable lipidic profile [8]. This could lead to potential hepatotoxicity.

Primate studies partially support findings in humans, with cholestasis and abnormal liver function found in individuals subjected to chronic transfusion involving DEHP-containing equipment [27]. Some liver abnormalities persisted for over two years post-transfusion in a study involving Rhesus monkeys [27], thus highlighting the potentially long-lasting effects of DEHP exposure caused by medical procedures on liver metabolism.

Studies on the hepatotoxicity of DEHP performed in rodents are less conclusive, with some reporting liver damage and dysfunction after dietary exposure in rats [28] and mice [29], while others report no liver injuries following DEHP exposure [30]. This is tied to the limitations of rodent models to recapitulate human hepatic pathophysiology. Notably, the effect of DEHP on peroxisome proliferation found in rodent is not seen in humans [31]. Moreover, murine models do not reflect the wide spectrum of histological features associated with MASLD [32]. This highlights the need for relevant and widely used liver models to study the effects of DEHP, and more generally phthalates, notably with regards to metabolic diseases.

At the cellular level, DEHP and its main metabolite MEHP can negatively impact hepatocytes through multiple pathways, namely PPAR alpha and gamma activation, proliferation of peroxisomes and induction of peroxisomal proteins, induction of non-peroxisomal metabolism proteins, suppression of apoptosis, oxidative DNA damage, and inhibition of gap junction intercellular communication [33]. DEHP and MEHP metabolism has also been tied to modifications of ATP-binding cassette efflux transporter expression in human HepG2 cells, with a potential for hepatotoxicity and a more global impact through modification of xenobiotic distribution [34]. Another consequence of DEHP/MEHP exposure on the liver involves the regulation of metabolic pathways such as lipid metabolism, thus leading to metabolic diseases such as MASLD [35].

## 2.2. DEHP is a metabolic disruptor involved in the development of MASLD

DEHP has been associated with a heightened prevalence of MASLD in a human adult cohort [36], and studies in both animal and cellular models have supported and shed light on this link.

Rats exposed to DEHP showed exacerbated MASLD with alterations of the expression of PPAR alpha and of SREBP-1C, the latter being a transcription factor involved in the control of lipogenic genes such as HMG-CoA reductase and fatty acid synthase [37]. Subsequent oxidation and inflammation in the liver thus link DEHP to a combinatory effect with high-fat diets (HFD) in the promotion of MASLD development. In zebrafish (*Danio rerio*), transcriptomic

analyses have shown that exposure to low levels of DEHP can impact hepatic expression of genes associated with MASLD through fatty acid metabolism, while co-exposure to DEHP and ethanol of larval stages fed a HFD induces progression of liver steatosis [24,25].

Research performed in hepatic cell lines have yielded complementary results, with the DEHP metabolite MEHP impacting fatty acid metabolism and increasing hepatocyte triglyceride content [38]. Also, steatotic WIF-B9 hepatocytes co-exposed to MEHP and ethanol led to cell death *via* oxidative DNA damage, which may indicate the progression of steatosis towards a steatohepatitis-like state [24].

Those elements all converge towards the implication of DEHP/MEHP in the onset and/or the pathological progression of MASLD, notably through modifications in the regulation of the expression of genes involved in lipid metabolism.

Considering the importance of cellular communication in the processes associated with MASLD, e.g., inflammation, cell death or fibrosis, studying the information carriers involved in those cellular interactions could improve our understanding and lead to better diagnostic options for this pathology. In this regard, EVs, known players of cellular communication and previously shown to be impacted by DEHP exposure, are prime candidates for investigation [39,40].

## 3. Extracellular vesicles and MASLD

EVs are biological particles delimited by a lipid-bilayer, emitted by nearly all cells and representative of their physiopathological status. EVs can be found in virtually every biological liquid, an element that, in association with the complexity of their content/cargo (lipids, proteins, mRNA, microRNA (miRNA), mitochondria, etc.) and their ability to target specific subsets of cells, empowers them with cellular communication capabilities and enables their utilisation as potential biomarkers [41,42].

In the liver, EVs of interest are mainly endosome-derived small EVs (30–150 nm diameter) and plasma membrane-derived large EVs (200–1000 nm diameter), that notably contribute to detoxification and regenerative hepatic dynamics [43]. Both total circulating EVs and hepatocyte-derived EVs are elevated in patients with MASLD [44–46].

As players of cellular communication within the liver, EVs can be associated with the onset and development of MASLD. Release of EVs by both hepatocytes and adipocytes following lipotoxic stress can lead to the targeted delivery of pro-inflammatory cargo to monocytes and macrophages [47,48].

Besides inflammation promoting mechanisms, EVs emitted by hepatocytes undergoing lipotoxic stress can also target endothelial and hepatic stellate cells (HSCs), leading to the activation of angiogenic and fibrogenic pathways [49,50].

EVs can also be associated with cross-talk phenomena between more than two types of cells, leading to a complex web of interactions with the potential to amplify and echo any dysregulation. This can be illustrated by the case of endothelial cells, the aforementioned targets of hepatocyte-derived EVs, which can in turn emit EVs that contribute to liver fibrosis [51].

Completing the histopathological elements associated with MASLD, EVs released by hepatocytes exposed to a lipid-rich environment were found *in vitro* to express TRAIL [52], an apoptotic ligand that could contribute to the increase in cell death typically found in the progression of liver steatosis towards MASH.

The biological effects of EVs released in the liver following exposure to lipogenic toxicants by promoting inflammation, cell death, immune modulation, fibrosis and angiogenesis, are likely involved in the progression of MASLD towards MASH. These
possible mechanisms deserve further study, especially in the context of exposure to pollutants. Should such roles for EVs be confirmed, a major goal would then be to identify the EV cargoes that mediate the intercellular communication that leads to MASH.

It is suspected that EVs can uptake cargo-like pollutants through endocytosis and direct fusion with the cell membrane but, to our knowledge, only one publication has so far reported that [53].

## 4. EV-derived miRNAs as putative biomarkers of DEHP exposure in MASLD

EVs contain stable and RNAse-resistant [54] miRNAs. These small and non-coding RNA molecules 20 to 25 nucleotides in length can alter the post-transcriptional regulation of proteins through binding to their corresponding mRNAs [55]. miRNAs are thus actors of intracellular processes through their regulatory nature, and their levels can reflect dysregulations and the alterations of cellular responses during pathogenesis. EV-derived miRNAs, like other EV cargoes, mirror the state of the emitting cell at the moment the EVs are produced. This implies the potential for a high level of miRNA diversity within EVs, notably through the presence of miRNA clusters [56].

### 4.1. Circulating or EV-derived miRNAs in MASLD

The EV-associated miRNA landscape is highly diverse and can vary depending on factors such as cell type, physiological or pathological conditions, and cellular stimuli [57].

In the context of MASLD, the involvement of miRNAs associated with EV emitted by hepatocytes undergoing lipotoxic events has been documented in recent years, with certain miRNAs shown to directly target the expression of genes involved in lipid metabolism (Table 1 and Fig. 1). Examples include the ASGR1⁺ EV-derived miR-128, increased in patients with MASLD [58]. Potential actors of interest also include miR-122 and miR-192, both found elevated in circulating EV-derived from hepatocytes undergoing MASLD [49,58,59]. miR-122 is particularly interesting, with increased

expression in serum EVs from mice on a HFD [60], as well as in patients with MASLD [58,61]. Furthermore, decreased liver expression of miR-122 has been linked to an increase in tissue-remodeling genes, such as hypoxia-inducible factor-1 alpha and vimentin in murine MASH-associated liver fibrosis [62]. In addition, serum levels of miR-21, miR-34, miR-122, miR-129, miR-135, miR-451 and miR-504 were increased in patients with MASLD, and as such could be useful biomarkers of the disease [60,61].

Hepatocyte-derived small EVs enriched with miR-192 have also been associated with macrophage activation and a subsequent increase in the pro-inflammatory cytokines IL-6 and TNF-alpha, notably through modulation of the Rictor/Akt/FoxO1 signalling pathway, in patients with MASLD [63]. Other circulating miRNAs, such as the inflammation-associated miR-21 and miR-34, elevated in obesity, have been associated with pathogenic factors in MASLD patients [61,64,65]. Moreover, repression of the miR-29 family members was found in experimental fibrosis induced by liver intoxication [66]. HFD in mice can also induce the expression of miR-9 and the transfer of miR-9 via lipotoxic hepatocyte-derived EVs can induce M1 polarization of macrophages in cellular and rodent MASLD models [67]. The potential roles of miR-133, miR-29 and miR-199 have been suggested in mediating the progression of liver fibrosis in murine hepatocytes [66,68,69]. However, none of these miRNAs have been found within EVs (Table 1).

In vivo studies in C57BL/6J mice and the Apoe⁻/⁻ mouse metabolic disease model verified that miR-128 and miR-148 regulate cholesterol/lipid and energy homeostasis contributing to metabolic syndrome [70]. They also regulate low-density lipoprotein (LDL) uptake and cholesterol efflux by controlling the expression of the LDL receptor and ABCA1 proteins in physiologically relevant cell types such as liver and macrophage-derived cells [70].

Clusters of miRNAs have also been identified as potential actors in the onset and development of MASLD. This is the case for the miR-17-92 cluster linked to apoptosis and proliferation [64], or the miR-23-27-24 cluster associated with fibrosis [71]. Other miRNAs, miR-379 and miR-375, have been associated with cholesterol lipotoxicity or glucose homeostasis leading to the development and

<table><caption>**Table 1**

Circulating miRNA biomarkers for MASLD.</caption><thead><tr><th>miRNA biomarker</th><th>circulating miRNA</th><th>miRNA present in EVs</th><th>Relevance</th><th>References</th></tr></thead><tbody><tr><td>miR-192</td><td>X</td><td>X</td><td rowspan="3">Cholesterol synthesis/trafficking</td><td>[58,63,113]</td></tr><tr><td>miR-128</td><td></td><td>X</td><td>[70]</td></tr><tr><td>miR-148</td><td></td><td></td><td>[70]</td></tr><tr><td>miR-29</td><td>X</td><td>X</td><td rowspan="7">Fibrosis/steatosis</td><td>[66]</td></tr><tr><td>miR-34</td><td>X</td><td></td><td>[61,65]</td></tr><tr><td>miR-122</td><td>X</td><td>X</td><td>[58,60–62,65,113,114]</td></tr><tr><td>miR-128</td><td></td><td>X</td><td>[58]</td></tr><tr><td>miR-129</td><td>X</td><td>X</td><td>[60]</td></tr><tr><td>miR-133</td><td>X</td><td></td><td>[68]</td></tr><tr><td>miR-135</td><td>X</td><td>X</td><td>[60]</td></tr><tr><td>miR-145</td><td>X</td><td></td><td></td><td>[61]</td></tr><tr><td>miR-199</td><td></td><td></td><td></td><td>[69]</td></tr><tr><td>miR-451</td><td></td><td></td><td></td><td>[61]</td></tr><tr><td>miR-504</td><td></td><td></td><td></td><td>[60]</td></tr><tr><td>miR-9</td><td>X</td><td></td><td rowspan="7">Inflammation</td><td>[67]</td></tr><tr><td>miR-19</td><td>X</td><td>X</td><td>[114]</td></tr><tr><td>miR-21</td><td>X</td><td></td><td>[61]</td></tr><tr><td>miR-15</td><td></td><td>X</td><td>[65]</td></tr><tr><td>miR-16</td><td>X</td><td></td><td>[65]</td></tr><tr><td>miR-122</td><td>X</td><td>X</td><td>[65]</td></tr><tr><td>miR-125</td><td>X</td><td>X</td><td>[114]</td></tr><tr><td>miR-379</td><td></td><td></td><td></td><td>[72]</td></tr><tr><td>miR-375</td><td>X</td><td></td><td>Glucose homeostasis</td><td>[114]</td></tr><tr><td>miR-17-92 cluster</td><td>X</td><td></td><td>Apoptosis</td><td>[64]</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Contextualization of EV-associated candidate biomarkers within the progression of MASLD. Selected miRNAs and proteins are presented in conjunction with key stages of liver disease and cellular dysregulations. EV, extracellular vesicle; MASLD, Metabolic dysfunction–Associated Steatotic Liver Disease; miRNAs, microRNAs. Created with Biorender.com.</figcaption></figure>

progression of MASLD [72].

## 4.2. EV-derived miRNAs in the context of DEHP exposure

miRNAs impacted by DEHP exposure have been previously documented (Table 2 and Fig. 1). miR-122 is the most abundant miRNA in hepatocytes (~70 % of expressed miRNAs) [73] and has been extensively studied as a liver toxicity biomarker [74]. This makes miR-122 a potential candidate for an association between DEHP exposure and MASLD.

Other notable DEHP-associated miRNAs include dose- responsive miRNAs whose expression was significantly altered in

mouse liver following DEHP exposure; this is the case for miR-182 and miR-378 which, interestingly, have been linked to the activation of PPAR alpha [75]. In addition, Oka et al. (2018) [76] proposed three liver miRNAs, miR-22, miR-409 and miR-543, as biomarkers of the genotoxicity of DEHP, due to its metabolite MEHP. Other experimental evidence linking MEHP and cholesterol efflux was associated with increased expression of certain miRNAs such as miR-16, miR-101, miR-362, miR-501, miR-532 or miR-542 [77]. But these miRNAs have not been identified as circulating or present within EVs in the context of MASLDs (Table 2).

Regarding EV-derived miRNAs, select urinary phenol and phthalate biomarkers are associated with altered expression of

<table><caption>**Table 2**
Circulating miRNA biomarkers for DEHP/MEHP exposure possibly involved in related MASLD.</caption><thead><tr><th>miRNA biomarker</th><th>circulating miRNA</th><th>miRNA present in EVs</th><th>Relevance</th><th>References</th></tr></thead><tbody><tr><td>miR-22</td><td></td><td></td><td rowspan="6">Impacted by DEHP</td><td>[76]</td></tr><tr><td>miR-125</td><td>X</td><td>X</td><td>[75,78]</td></tr><tr><td>miR-182</td><td></td><td></td><td>[75]</td></tr><tr><td>miR-378</td><td></td><td></td><td>[75]</td></tr><tr><td>miR-409</td><td></td><td></td><td>[76]</td></tr><tr><td>miR-543</td><td></td><td></td><td>[76]</td></tr><tr><td>has-let-7</td><td>X</td><td>X</td><td></td><td>[78]</td></tr><tr><td>miR-15</td><td>X</td><td>X</td><td rowspan="12">Impacted by MEHP</td><td>[78]</td></tr><tr><td>miR-16</td><td>X</td><td></td><td>[77]</td></tr><tr><td>miR-19</td><td>X</td><td>X</td><td>[78]</td></tr><tr><td>miR-24</td><td>X</td><td>X</td><td>[78]</td></tr><tr><td>miR-29</td><td>X</td><td>X</td><td>[79]</td></tr><tr><td>miR-101</td><td></td><td></td><td>[77]</td></tr><tr><td>miR-106</td><td>X</td><td>X</td><td>[78]</td></tr><tr><td>miR-362</td><td></td><td></td><td>[77]</td></tr><tr><td>miR-374</td><td>X</td><td>X</td><td>[78]</td></tr><tr><td>miR-375</td><td>X</td><td>X</td><td>[78]</td></tr><tr><td>miR-501</td><td></td><td></td><td>[77]</td></tr><tr><td>miR-532</td><td></td><td></td><td>[77]</td></tr><tr><td>miR-542</td><td></td><td></td><td>[77]</td></tr></tbody></table>
eight EV-miRNAs in follicular fluid, hsa-let-7, miR-375, miR-24, miR-19, miR-125, miR-106, miR-374, and miR-15 [78]. Lastly, a positive correlation between urinary MEHP concentrations and EV-derived miR-29 expression levels was found in non-diabetic pregnant Mexican women; this association was not observed in gestational diabetes mellitus [79].

## 5. EV proteins as putative biomarkers of DEHP exposure in MASLD

Although EV-associated proteins can reach a high degree of diversity, their impact on regulatory pathways within the realm of EV-based cellular communication is less straightforward than that of miRNAs. Given the essence of EVs as instantaneous snap shots of the emitting cells, the magnitude of the impact of protein cargo on the receiving cells is bound to differ from emitting cells, both in effect and intensity [80]. Proteins of interest presented below and displayed in Table 3, mostly cover actors causally linked to the hepatocellular aspects of MASLD.

### 5.1. EV proteins in MASLD

Proteins associated with MASLD can be associated to multiple aspects and stages of its pathogenesis, and are thus implicated in a wide range of cellular functions.

Some of the proteins of interest are associated with steatosis. Cytochrome P450 family member CYP2E1 and receptor ASGR1/2 (asiaglycoprotein receptors 1 and 2) have thus been identified as present in EVs and directly linked with the state of hepatic health in MASLD patients [44–46]. Among hepatocyte-specific markers in circulating EVs, the levels of bile acyl-coenzyme A synthetase or fatty acid transport protein 5 (SLC27A5 or FATP5) are 3-4-fold greater in serum from patients with MASH compared to healthy controls [46,81]. Hepatocyte-derived EVs are also enriched in vanin-1, an ectoenzyme involved in cell adherence and migration, which plays a role in the pathogenesis of MASLD [59,82].

Other proteins circulating or present in EVs and suspected to play a role in MASLD are involved in lipid dysregulations. These

include circulating ApoE whose plasmatic level has been associated with MASLD [83], and circulating ApoA5 which has been shown to directly contribute to the pathogenesis of MASLD and is a marker for the increase in triglyceride accumulation in lipid droplets [84]. Furthermore, human livers with MASLD have been found to express an altered form of circulating ApoA5 [85]. Other candidates associated with lipid metabolism are already used as NALFD biomarkers. This is the case of Paraoxonase 1, an antioxidant circulating biomarker identified through a meta-analysis [86]. Serum concentration levels of another member of the paraoxonase family, Paraoxonase 3, have been associated with fatty liver index in a clinical study [87]. Signalling molecules have also been associated with lipid dysregulations and hepatic disease, with overexpression of the adaptor protein SHC1 linked to hepatocyte senescence and promotion of liver steatosis [88]. However these proteins remain to be identified as present into EVs (Table 3).

In line with the clinical progression of MASLD, multiple proteins linked to inflammation have been identified. Thus chemokines, such as CXCL10, have been identified as potential therapeutic targets [89], while the apoptotic ligand TRAIL, associated with pro-inflammatory effects through macrophages, has been detected in hepatocyte-derived EV secreted under lipotoxic conditions [52]. Other key players in the development of hepatic inflammation, such as acid protease cathepsin D, have been associated with the EV proteome in an experimental model of fatty liver disease [90]. Cathepsin D seems of particular interest since it can be tied to three hallmarks of MASLD: lipid metabolism dysregulations, inflammation, and cell death. Another candidate of particular interest at the crossroad between apoptosis and lipid metabolism is the chaperone clusterin, whose hepatocyte-specific overexpression has been described as attenuating diet-induced MASH [91].

Both ceramide and sphingosine 1-phosphate (S1P) are involved in endosomal EV formation [92] and also implicated in the pathogenesis and progression of MASH, with concurrent upregulation of sphingosine kinase 1 (SK1) [93]. Palmitate-induced lipotoxic EVs are enriched in S1P through SK1 [48]. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor; this signaling axis for macrophage infiltration

<table><caption>**Table 3**
Putative EV protein biomarkers for MASLD and/or impacted by DEHP.</caption><thead><tr><th>Protein biomarker</th><th>Alias</th><th>Present in EV</th><th>Impacted by DEHP</th><th>Relevance</th><th>References</th></tr></thead><tbody><tr><td>CYP2E1</td><td>Cytochrome P450 Family 2 Subfamily E Member 1</td><td>X</td><td></td><td>Liver metabolism</td><td>[44]</td></tr><tr><td>ASGR1/2</td><td>Asialoglycoprotein Receptor 1/2</td><td>X</td><td></td><td>Lipid metabolism</td><td>[44–46]</td></tr><tr><td>SLC7A5 (FATP5)</td><td>Solute carrier family 27A5 (fatty acid transport protein 5)</td><td>X</td><td></td><td></td><td>[46,81]</td></tr><tr><td>VANIN-1</td><td>Vascular non-inflammatory molecule-1</td><td>X</td><td></td><td></td><td>[59,82]</td></tr><tr><td>ApoE</td><td>Apolipoprotein E</td><td></td><td></td><td></td><td>[83]</td></tr><tr><td>ApoA5</td><td>Apolipoprotein A5</td><td></td><td></td><td></td><td>[83,85]</td></tr><tr><td>PON1</td><td>Paraoxonase 1</td><td></td><td></td><td></td><td>[86]</td></tr><tr><td>PON3</td><td>Paraoxonase 3</td><td></td><td></td><td></td><td>[87]</td></tr><tr><td>CLU</td><td>Clusterin</td><td></td><td></td><td></td><td>[91]</td></tr><tr><td>SREB-2</td><td>Sterol regulatory element binding protein-2</td><td></td><td>X</td><td></td><td>[112]</td></tr><tr><td>PPAR</td><td>Peroxisome proliferator-activated receptor</td><td></td><td>X</td><td></td><td>[108]</td></tr><tr><td>TRAIL</td><td>TNF Superfamily Member 10</td><td>X</td><td></td><td>Inflammation</td><td>[52]</td></tr><tr><td>MMP-3</td><td>Matrix Metalloproteinase 3</td><td>X</td><td>X</td><td></td><td>[99,100]</td></tr><tr><td>ITGβ<sub>1</sub></td><td>Integrin β<sub>1</sub></td><td>X</td><td></td><td></td><td>[47]</td></tr><tr><td>CXCL10</td><td>C-X-C Motif Chemokine Ligand 10</td><td>X</td><td></td><td></td><td>[89,98]</td></tr><tr><td>CTSD</td><td>Cathepsin D</td><td></td><td></td><td></td><td>[90]</td></tr><tr><td>VTN</td><td>Vitronectin</td><td></td><td></td><td></td><td>[94]</td></tr><tr><td>TF</td><td>Tissue factor</td><td>X</td><td>X</td><td>Fibrosis</td><td>[102]</td></tr><tr><td>MMP-2</td><td>Matrix Metalloproteinase 2</td><td>X</td><td></td><td></td><td>[97]</td></tr><tr><td>MMP-9</td><td>Matrix Metalloproteinase 9</td><td>X</td><td></td><td></td><td>[97]</td></tr><tr><td>SK1</td><td>Sphingosine kinase 1</td><td>X</td><td></td><td></td><td>[48,107]</td></tr><tr><td>CCN2</td><td>Connective tissue growth factor</td><td>X</td><td></td><td></td><td>[95]</td></tr><tr><td>MMP-10</td><td>Matrix Metalloproteinase 10</td><td></td><td>X</td><td></td><td>[99,101]</td></tr><tr><td>MMP-14</td><td>Matrix Metalloproteinase 14</td><td></td><td>X</td><td></td><td>[99]</td></tr></tbody></table>
during hepatic lipotoxicity is a potential therapeutic target for MASH.

Finally, proteins associated with fibrosis, characterized by excess deposition of a collagen-rich extracellular matrix, have also been reported in EVs. A deficiency in the glycoprotein vitronectin has been linked to an attenuation of hepatic fibrosis in a MASH mouse model [94]. Moreover, hepatic stellar activated cell-derived small EVs contain CCN2 (connective tissue growth factor), an important pro-fibrogenic molecule [95]. Metalloproteinases (MMPs) are crucial to extracellular matrix degradation as well as processing of growth factors, cytokines and surface transmembrane molecules. Interestingly, proteomics have shown the presence of secreted-type (soluble) MMPs (MMP-1, -13, -2, -9, -3, -10, -7, -19) in EV-derived from various cell types and isolated from various biological fluids (blood plasma, malignant ascites, urine, saliva, and synovial fluid) [96,97].

The aforementioned proteins have been described as part of the EV proteome, as is the case for CXCL10 [89,98] or ASGR1 [44–46], making them prime candidates for EV-based biomarkers in MASLD.

## 5.2. EV proteins associated with DEHP exposure

DEHP and its metabolite MEHP have been associated with modifications of protein expression in various cell types. Interestingly, multiple studies highlight an impact of these pollutants on the expression of proteins associated with extracellular matrix regulation, a key element whose unbalance can lead to fibrosis [36], a well-known hallmark in the progression of MASLD.

DEHP was thus found to induce the production of inflammatory molecules in human macrophages, causing, notably, an increase in the expression of the matrix metalloproteinases MMP3, MMP10 and MMP14 [99], enzymes whose presence in small EVs and microparticles has been ascertained [100,101]. MMP-2 and MMP-9, as well as TIMP-2, have also been shown to be present in EVs [96]. The action of DEHP/MEHP on MMPs might, however, be more complex than the simple modulation of their expression. MMP activity and the MMP/TIMP (endogenous MMP inhibitor) balance should also be considered [96].

DEHP, added to inflammatory macrophages, was also shown to induce the release of tissue factor (TF) into large EVs. TF being a primary cofactor involved in activation of the coagulation pathway required for liver inflammation and fibrosis, this action of DEHP appears specifically linked to the inflammatory state of the cells [102].

# 6. EV lipids as putative biomarkers of DEHP exposure in MASLD

Diversity in the origin of EVs at the subcellular level leads to differences in their lipid composition. The lipid content of hepatic EVs can be expected to match that of the endosomal or plasma membranes, important sources of EVs in the liver. Considering the importance of lipid metabolism dysregulations in the onset and development of MASLD, the composition of the lipid bilayers in hepatic EVs could prove informative. Furthermore, with their forefront position in the interaction between EVs and their target cells, lipids could also prove to be important actors in MASLD as mediators of cellular communication.

## 6.1. EV lipids in MASLD

The accumulation of saturated free fatty acids, free cholesterol and sphingolipids such as ceramides is associated with the progression of MASLD [103]. EVs are known to carry a diverse range of lipids, including phospholipids, cholesterol, sphingolipids, and

glycerolipids. These lipids can reflect the metabolic state of cells and tissues, making them attractive candidates for biomarker discovery in MASLD. EV-derived lipids offer a window onto the lipid metabolism of the emitting cells that complement the quantitative and qualitative analysis of hepatic EVs.

From a mechanistic standpoint, hyperlipidemia is deeply tied to the production of EVs in hepatocytes through the involvement of multiple pathways. Notably, the accumulation of saturated free fatty acids is associated with the activation of the DR5/caspase8/caspase3/ROCK1 pathway [104], which mediates small EV production [52]. Up-regulation of the MLK3/JNK [105] pathway, by ongoing lipid accumulation, is also linked with an increase in small EV production. Potentially completing those elements, lipotoxic stress sensing through the inositol requiring enzyme IRE1 alpha, could also be tied to an increase in small EV release [106].

Alterations of EV production through those molecular cascades have consequences on the hepatic environment as a result of the biological activity of EV cargoes; these can contribute to the development of MASLD as was described above for miRNAs and proteins. Lipidomic approaches to investigate EV contents within the paradigm of MASLD have yielded what seem to be promising results. In a study following the lipid content of hepatic EVs pre- and post-bariatric surgery of MASLD patients, the authors have shown the ability for lipidomics focussed on sphingolipids to correlate lipid ratios to the benchmark histologic MASLD activity score (MAS), currently in use for the assessment of biopsies [44]. Accordingly, lipid contents of small EVs are considered as witnesses of the metabolic pathway shifts that occur during the pathological progression of MASLD, thereby allowing a resolutive view of the progression of the disease.

The interest in sphingolipids does not however stop here, as S1P from EV produced by immortalised mouse hepatocytes after lipotoxic stress was found to be chemoattractive to macrophages [48,107]. This discovery underlies a potential role of EV lipids in the establishment of chemotactic gradients leading to the development of the inflammatory landscape associated with the progression of MASLD. Interestingly, S1P is also associated with an increase in small EV production in hepatocytes exposed to lipotoxicity [48].

## 6.2. Effect of DEHP on lipids

Studies have suggested that exposure to DEHP can lead to modifications in the lipid content, both in tissues and biological fluids. Notably, peroxisome proliferator-activated receptor (PPAR)-alpha gene expression is up-regulated in MEHP-treated mice [108]. It is known that PPARs play a role in intracellular lipid metabolism by up-regulating the expression of enzymes involved in the conversion of fatty acids into acyl-coenzyme A esters, such as acyl coenzyme A synthetase. On the other hand, DEHP is associated with decreased sphingomyelin levels in rat livers [109]. Moreover, administration of clofibrate and DEHP, altogether ligands of PPAR, to rats causes liver enlargement, peroxisome proliferation and induction of enzymes of both peroxisomal and microsomal fatty acid oxidation [110]. Furthermore, inhibition of sterol synthesis is also observed in the liver of DEHP-fed rabbits [111]. DEHP exposure also affects lipid metabolism through up-regulation of SREBP-2 mRNA expression and protein levels in the liver of rats, indicating that DEHP can increase the synthesis of cholesterol [112]. However these proteins linked to lipid metabolism remain to be identified as present within EVs (Table 3).

While it is not impossible that DEHP could specifically modify the lipid content of EVs, it seems more fitting to consider that changes in EV content reflect changes in the state of the emitting cell. Accordingly, it seems far more informative to consider the EV lipid content through lipidomic approaches, such as the ones
mentioned earlier, and to study variations within those profiles in
response to DEHP exposure.

7. Use of those candidates as biomarkers

While the complexity of the EV-associated miRNA landscape is
considerable, the use of those molecules as biomarkers is undeni-
ably of high interest and potential. Characterizing the EV-associated
miRNA landscape has significant implications for understanding
intercellular communication, biomarker discovery, and the devel-
opment of diagnostic and therapeutic strategies.

Once a highly resolutive understanding of the panel of miRNAs
associated with risk factors (e.g., pollutants such as EDs) for the
onset and development of MASLD is achieved, the complexity of
those signatures will entail a high degree of precision in diagnosis.
This will enable an understanding not only of the regulatory state of
the emitting cells, but also of the impact of the targeted EV-derived
miRNAs on the recipient cells. We have reported in Table 1 the
putative EV miRNA biomarkers of MASLD characterized by the
accumulation of fat in the liver, which can progress to inflammation
and liver damage such as fibrosis. In particular, circulating EVs from
diet-induced MASLD/MASH mice were enriched in miRNA-122 and
miR-192 - two well-investigated and abundant microRNAs in he-
patocytes [62,113]. In human, miR-122 (which constitutes more
than 70 % of the total liver miRNA pool) and miR-192 (upregulated
by TGFβ1) were significantly elevated in serum of patients with
MASH compared to those of patients with simple steatosis
[65,114,115].

Regarding more specifically DEHP, several EV-related miRNAs
might be putative biomarkers of exposure and/or effect in the
context of MASLD. Indeed, urinary concentrations of DEHP and its
metabolites are correlated with the expression of EV-miRNAs iso-
lated from follicular fluid in women undergoing *in vitro* fertilization
[78].

Regarding their use as biomarkers, selected EV-associated pro-
teins could prove to be one of the simplest tools to ascertain
diagnosis and evolution of MASLD in a global fashion. Considering
how widespread are assays to quantify proteins of interest, the
selection of a few suitable candidates or the consideration of ratios
between suspected candidates could be the first step towards
quickly deciphering the hepatic health of a patient based on EV
content. In line with this, as illustrated in Table 3, one might pro-
pose a panel of proteins that deserve further attention, especially in
the context of MASLD patients possibly exposed to DEHP. Recent
research has explored the potential of ASGR1 as a biomarker for
MASLD. It provides a specific marker for small EVs of hepatic origin
that may be useful to discriminate and purify hepatocyte-derived
small EVs in complex biological samples like blood in which a
mixed population of small EV secreted by different cell types are
present [116]. Similarly, SLC27A5 and ASGR1 may be liver specific
markers of EVs from steatotic hepatocytes in serum from patients
with MASLD [45,46,81].

Selected miRNAs and proteins are presented in Fig. 1, in
conjunction with key phases of liver health, and cellular
dysregulations.

As mentioned earlier, one of the main strengths of lipid-based
EV biomarkers in MASLD is their direct tie to the metabolic basis
of the pathology. While miRNAs and proteins can inform on cellular
communication pathways susceptible to spark or reinforce in-
flammatory processes, lipids can provide information on the lipid
metabolism dysregulations ongoing in hepatic cells. In this sense,
lipid biomarkers could offer a more resolutive approach to MASLD
diagnosis and monitoring than miRNA and protein-based assays.

8. Clinical applications in liver diseases

Although EVs have potential clinical value in various liver dis-
eases, the field is still in the pre- or early clinical stage and there are
many challenges to overcome before using EVs clinically [44,117].

Challenges associated with the use of EVs as biomarkers in a
clinical setting are tied to their quantity and the complexity of
biological samples. Physical characterization is currently heavily
reliant on electron microscopy, while enrichment is performed
mostly using ultracentrifugation, with both techniques using large
instrumentation with long preparation times. New techniques are
being developed and implemented in research facilities, with
physical characterization techniques such as Single EV Analysis
Method and enrichment techniques such as Microfluidic Filtering
or Contact-Free Sorting. Both techniques could prove powerful
allies for biomarker-based clinical analyses [118]. Envisioned clin-
ical applications of EVs reflect the width of this field of study, as
emphasized by the ever growing number of publications. From
therapy using EVs as targeted carriers for active compounds to
reach specific groups of cells or well-protected structures, to
prognosis and use as biomarkers, creative solutions harnessing the
peculiarities of EVs are on the rise.

Liver-derived EV enrichment in biological fluids will be of major
importance in order to harness representative and thus useful
material in the context of MASLD. This could be achieved through
targeting of hepatocyte-specific receptors found in hepatocyte-
derived EVs such as the asialoglycoprotein receptor ASGR [45,58],
with the concept being extendable to other hepatic cell types [117].
Molecular answers to the conundrum that is MASLD are being
developed, improvements in imagery [119] could also play a part in
the creation of an efficient and painless toolset for practitioners to
use. This would complement other tools in development such as
screening indicators based on index combinations, namely the
triglyceride-glucose, body-mass and waist-circumference indexes
[120]. With the rising diversity in the clinical uses of EVs, techno-
logical advances in one field are bound to benefit others, height-
ening the chances that EV-based biomarkers become a standard -
reliable and stream-lined - solution for the diagnosis and moni-
toring of MASLD amongst other pathologies.

9. Conclusion

This short review aimed to highlight the relevance of EVs in
helping the diagnosis and monitoring of chronic liver pathologies
for which gold standards are lacking. The application of this
concept to MASLD - more particularly in order to ascertain and
follow the effects of endocrine disruptors such as DEHP on the
disease - seems highly promising granted that technological ad-
vances allow for efficient analysis of EV contents in the clinical
setting. Reliable biomarkers, whose presence and relative amount
can be precisely tied with histopathological features, will be the
cornerstone of any such application; further work to characterize
such molecular markers is essential to help withstand the preva-
lence and growth of MASLD.

While this review focused on DEHP and its metabolite MEHP,
growing evidence connects other endocrine disrupting com-
pounds, notably those causing hepatotoxicity, with changes in
miRNA, protein and lipid contents of the exposed cells or EVs
thereof [121,122]. This therefore opens numerous possibilities
regarding the use of EV-based information for research or clinical
purposes. The strategies presented here, on the potential of EV-
based biomarkers to monitor MASLD associated to DEHP expo-
sure, could be extended to other xenobiotics such as members of
the polycyclic aromatic hydrocarbon family given their ability to
induce oxidative stress in hepatocytes through EVs [40] and their
documented links with MASLD [123] and more general obesogenic mechanisms [6].

## Funding

This work is supported by Oberon, a collaborative project funded by the EU Framework Programme for Research and Innovation Action, Horizon 2020, under grant agreement n°825712, and by our institutions, Inserm and Université de Rennes.

## CRediT authorship contribution statement

**Pierre-Etienne Merret:** Writing – original draft. **Lydie Sparfel:** Writing – original draft, Writing – review & editing. **Catherine Lavau:** Writing – review & editing. **Dominique Lagadic-Gossmann:** Conceptualization, Funding acquisition, Writing – review & editing. **Corinne Martin-Chouly:** Conceptualization, Writing – original draft, Writing – review & editing.

## Declaration of competing interest

All authors declare no conflict of interest.

## Acknowledgments

The figure was prepared using the free version of the Biorender software.

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