# Lack of effects of polystyrene micro- and nanoplastics on activity and expression of human drug transporters

Valentin Tastet, Marc Le Vée, Alexis Verger, Nolwenn Brandhonneur, Arnaud Bruyère, Olivier Fardel

► To cite this version:

Valentin Tastet, Marc Le Vée, Alexis Verger, Nolwenn Brandhonneur, Arnaud Bruyère, et al.. Lack of effects of polystyrene micro- and nanoplastics on activity and expression of human drug transporters. Environmental Toxicology and Pharmacology, 2024, 111, pp.104563. 10.1016/j.etap.2024.104563. hal-04717934

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# Lack of effects of polystyrene micro- and nanoplastics on activity and expression of human drug transporters

Valentin Tastet<sup>a</sup>, Marc Le Vée<sup>a</sup>, Alexis Verger<sup>b</sup>, Nolwenn Brandhonneur<sup>b</sup>, Arnaud Bruyère<sup>a</sup>, Olivier Fardel<sup>c,*</sup>

## ARTICLE INFO

Edited by Malcolm D. Tingle

### Keywords:

- ABC transporters

- Hepatic HepaRG cells

- Intestinal Caco-2 cells

- Plastic particles

- Polystyrene

- Solute carriers

## ABSTRACT

Micro- and nanoplastics (MPs/NPs) constitute emerging and widely-distributed environmental contaminants to which humans are highly exposed. They possibly represent a threat for human health. In order to identify cellular/molecular targets for these plastic particles, we have analysed the effects of exposure to manufactured polystyrene (PS) MPs and NPs on *in vitro* activity and expression of human membrane drug transporters, known to interact with chemical pollutants. PS MPs and NPs, used at various concentrations (1, 10 or 100 µg/mL), failed to inhibit efflux activities of the ATP-binding cassette (ABC) transporters P-glycoprotein, MRPs and BCRP in ABC transporter-expressing cells. Furthermore, PS particles did not impair the transport of P-glycoprotein or BCRP substrates across intestinal Caco-2 cell monolayers. Uptake activities of solute carriers (SLCs) such as OCT1 and OCT2 (handling organic cations) or OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 (handling organic anions) were additionally not altered by PS MPs/NPs in HEK-293 cells overexpressing these SLCs. mRNA expression of ABC transporters and of the SLCs OCT1 and OATP2B1 in Caco-2 cells and human hepatic HepaRG cells were finally not impaired by a 48-h exposure to MPs/NPs. Altogether, these data indicate that human drug transporters are unlikely to be direct and univocal targets for synthetic PS MPs/NPs.

## 1. Introduction

Plastic particles, comprising nanoplastics (NPs) (size<1 µm) and microplastics (MPs) (1 µm<size<5 µm) (Gigault et al., 2018), are widely present in the environment, including soils, water, air, food and indoor atmosphere (Ageel et al., 2022; Kek et al., 2023; Lehner et al., 2019). Humans worldwide are consequently largely exposed to these emerging pollutants, which may prove harmful towards health (Bouwmeester et al., 2015; Chang et al., 2020; Xu et al., 2022; Zarus et al., 2021). Identification of possible human cellular and molecular targets for NPs/MPs is therefore an important issue (Ali et al., 2023; Hu and Palić, 2020; Khan and Jia, 2023; Ali et al., 2023), which currently warrants the growing number of studies (Ge et al., 2023; Koner et al., 2023; Lin et al., 2022; Sun et al., 2023; Xu et al., 2022). In this context, transport across membrane barriers has been proposed as possibly impaired by plastic particles (Liang et al., 2021). In particular, ingested MPs/NPs may damage the intestinal barrier (Huang et al., 2021), notably by exerting toxicity towards intestinal epithelial cells (Cui et al., 2023; DeLoid et al.,

2021; Liu et al., 2020).

Membrane drug transporters belonging to the ATP-binding cassette (ABC) membrane protein family or to the solute carrier (SLC) family are key components of biological barriers, especially at the intestine, liver, blood-brain and kidney level (Giacomini et al., 2010). ABC transporters such as P-glycoprotein (P-gp), encoded by the multidrug resistance gene 1 (MDR1/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2), act as efflux proteins, thereby decreasing accumulation of drugs into cells, preventing their passage across blood-tissue barriers and mediating their secretion into the bile or the urine (Choi and Yu, 2014). SLCs usually behave as uptake transporters of organic cations or anions into cells, notably into hepatocytes and kidney proximal tubular cells (Liu, 2019). Drug transporters thus play a major role in pharmacokinetics and are implicated in drug-drug-interactions and drug toxicity (Petzinger and Geyer, 2006). Beyond drugs, environmental chemicals have been shown to interact with drug transporters (Guéniche et al., 2020; Michel et al., 2023; Nicklisch and Hamdoun, 2020). Various plastic-associated

<footnote><sup>a</sup> Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, Rennes 35000, France</footnote>

<footnote><sup>b</sup> Univ Rennes, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, Rennes 35000, France</footnote>

<footnote><sup>c</sup> Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, Rennes 35000, France</footnote>

<footnote>* Correspondence to: Irset, Campus Santé, 2 Avenue Pr Léon Bernard, Rennes 35000, France.
E-mail address: olivier.fardel@univ-rennes1.fr (O. Fardel).</footnote>
chemicals, including flame retardants and bisphenols, can thus inhibit
transporter activities, be their substrates or regulate their expression
(Rosellini et al., 2023; Tastet et al., 2023a). Plastic particles themselves
have been hypothesized to decrease ABC transporter activity, notably in
human Caco-2 cells, even if the resulting increase of ABC transporter
substrate accumulation is rather modest (by approximately 20 %) (Wu
et al., 2019). This may however result in enhanced sensitivity to pol-
lutants substrates for ABC efflux pumps, such as arsenic (Chen et al.,
2023). Plastic particles can also modulate the expression of ABC trans-
porters (Paul et al., 2023). The exact nature of transporters, including
ABC transporters, interacting with MPs/NPs remains however not
characterized (Wu et al., 2019). Against this backdrop, the present study
was designed to carefully investigate the effects of an exposure to syn-
thetically manufactured polystyrene (PS) MPs and NPs used at various
concentrations on activities and expressions of main human ABC and
SLC transporters implicated in pharmacokinetics. Our data showcase PS
particles do not interact with transporter activities or expressions in
cultured transporter-expressing cells, suggesting that drug transporters
are unlikely to be direct and univocal major targets of synthetic PS MPs
and NPs at the concentrations (up to 100 µg/mL) used in the study.

**2. Material and methods**

**2.1. PS particles and chemicals**

Spheric PS MPs (native mean size 5.12 µm, reference PP-50-10) and NPs (native mean size 50 nm, Polybead® Polystyrene Microsphere reference 08691-10), suspended in water, were commercially provided by Spherotech (Lake Forest, IL, USA) and CliniSciences (Nanterre, France), respectively. Suppliers for chemicals are indicated in Table S1.

**2.2. PS particle size characterization**

Size distributions of PS MPs and NPs were characterized by light
diffraction using a Mastersizer S™ (Malvern Instruments, Orsay, France)
and by dynamic light scattering using a Malvern Zetasizer nano ZS™
(Malvern Instruments), respectively, as previously described (Huang
et al., 2023; Wu et al., 2019).

**2.3. Cell culture**

Human ABC transporters-expressing cells, i.e., P-gp-positive MCF7R
cells, MRPs-expressing HuH-7 cells and BCRP-transfected HEK-293 cells
(HEK-BCRP cells), were cultured in Dulbecco's modified Eagle medium
(DMEM) (Life Technologies, Saint-Aubin, France), supplemented with
10% (v/v) fetal calf serum (FCS) and antibiotics (100 IU/mL penicillin
and 100 µg/mL streptomycin for MCF7R cells and HuH-7 cells or 100
IU/mL amoxicillin, 100 µg/mL erythromycin and 2 mg/mL G418 for
HEK-BCRP cells), as already reported (Chedik et al., 2017). Human
HEK-293 cells expressing human SLC transporters, i.e., organic cation
transporter (OCT) 1/SLC22A1 (HEK-OCT1 cells), OCT2/SLC22A2
(HEK-OCT2 cells), organic anion transporter (OAT) 1/SLC22A6
(HEK-OAT1 cells), OAT3/SLC22A8 (HEK-OAT3 cells), organic anion
transporting polypeptide/OATP 1B1/SLCO1B1 (HEK-OATP1B1 cells),
OATP1B3/SLCO1B3 (HEK-OATP1B3 cells) or OATP2B1/SLCO2B1, as
well as control HEK-MOCK cells were maintained in DMEM supple-
mented with 10% (vol/vol) FCS, 1% (vol/vol) MEM non-essential
amino acids solution (Life Technologies, Villebon-Sur-Yvette, France),
1 µg/mL bovine insulin, 20 IU/mL penicillin, and 20 µg/mL strepto-
mycin. Human hepatoma HepaRG cells were cultured in Williams' E
medium supplemented with 10% (vol/vol) FCS, 100 IU/mL penicillin,
100 µg/mL streptomycin, 5 µg/mL insulin, 2 mM glutamine, and 50 µM
hydrocortisone hemisuccinate, as previously described (Le Vee et al.,
2013); their full hepatocytic differentiation was induced by adding 2% (vol/vol) dimethylsulfoxide (DMSO) for 2 weeks in the culture medium.
Human intestinal Caco-2 cells were routinely cultured in DMEM

supplemented with 20 IU/mL penicillin, 20 µg/mL streptomycin and
10 % (vol/vol) FCS. For transport assay, Caco-2 cells were seeded at 12
500 cells/cm² in Falcon® Cell Culture Inserts on polyethylene tere-
phthalate membranes (0.4 µm pore size, 6.4 mm diameter), purchased
from Dutscher (Bernolsheim, France). Culture media were changed
three times a week, and cells were used on Days 21-22 post-seeding, as
previously reported (Guéniche et al., 2023).

**2.4. PS particle treatment**

Three concentrations of PS MPs or NPs, i.e., 1, 10 and/or 100 µg/mL, were investigated in the study. The low concentration of 1 µg/mL is in the range of plastic particle concentrations reported in human blood, with a mean of total plastic particles in blood samples of 1.6 µg/mL (Leslie et al., 2022); this suggests that this 1 µg/mL concentration is environmentally-relevant. The higher PS particle concentrations of 10 µg/mL may be compatible with intraluminal gut concentrations of ingested plastic particles. Indeed, the daily intake of MPs for adults has been estimated to be 40.1 µg/kg body weight (Zuccarello et al., 2019), which corresponds to a total intake of 2807 µg MPs for a 70 kg-subject; with the hypothesis that this intake occurs via one meal and considering an intestinal luminal volume of 250 mL (Giacomini et al., 2010; Tastet et al., 2023b), this results in an estimated intraluminal gut MP concentration of 11.2 µg/mL. Finally the concentration of 100 µg/mL, although rather high, remains in the range of those previously commonly used in *in vitro* toxicological studies with MPs and NPs (Ruan et al., 2023; Sae-nen et al., 2023).

**2.5. Cell viability assay**

Cell viability was analyzed through light microscopic examination of
the culture and through the use of the yellow tetrazolium salt (MTT)
assay, as previously described (Carmichael et al., 1987).

**2.6. Drug transporter assays**

Fluorescent substrates and reference inhibitors were used to evaluate
the effects of PS particles on SLC and ABC transporter activities by
measuring cellular accumulation or retention of fluorescent reference
substrates in transporter-overexpressing cells in the absence (control
condition) or presence of PS particles or specific inhibitors. The incu-
bation times, analytical methods, transporter-overexpressing cells and
reference substrates and inhibitors used for these transport assays have
been previously validated (Chedik et al., 2017; Tastet et al., 2023b) and
are summarized in Table S2. Transporter activity assays were performed
in a transport assay medium composed of Hank's balanced salt solution
(HBSS) supplemented with 10% (vol/vol) FCS. Intracellular accumu-
lation or retention of substrates were determined by spectrofluorimetry.
Data were normalized to protein content and were expressed as per-
centages of substrate concentration found in control cells not exposed to
PS particles or reference inhibitors (for accumulation assays) or as
percentages of initial substrate loading (for retention assays).

**2.7. Caco-2 cell monolayer integrity assays**

Integrity of the Caco-2 cell monolayer on culture inserts was
routinely analyzed through trans-epithelial electrical resistance (TEER)
measurements using a Millipore Millicell® ERS-2 Volt-Ohm meter sup-
plied by Merck (Darmstadt, Germany) and by measuring paracellular
passage of Lucifer Yellow, as previously reported (Vila et al., 2018).

**2.8. Transport assays across Caco-2 cell monolayer**

Transport of [³H]-estrone-3-sulfate (E3S) and rhodamine 123 across
Caco-2 cell monolayers, established in culture inserts, was performed as
previously described (Guéniche et al., 2023). Caco-2 cell monolayers
A

<figure><img src="image_2.png" /></figure>

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

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

B

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

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

**Fig. 1.** Effects of MPs/NPs on human ABC transporter activities. (A) Cellular accumulation or retention of ABC transporter substrates, i.e., rhodamine 123 (for P-gp-related activity assay), carboxy-2',7'-dichlorofluorescein (CDCF) (for MRPs-related activity assay) and Hoechst 33342 (for BCRP-related activity assay), were determined in the absence (control) or presence of MPs/NPs used at 1, 10 or 100 µg/mL or of the transporter inhibitors cyclosporin A (CsA, 100 µM), probenecid (2 mM) or fumitremorgin C (FTC, 10 µM). Data are expressed as percentages of cellular substrate accumulation found in control cells (for rhodamine 123 accumulation assays) or as percentages of initial substrate loading (for CDCF and Hoechst 33342 retention assays). They are the means ± SD of at least 4 independent assays. Dashed lines indicate levels of substrates in control cells. **p**<0.0001 when compared to control cells. (B) Efflux ratio of the P-gp substrate rhodamine 123 and the BCRP substrate E3S across Caco-2 cell monolayers not exposed (control) or previously exposed to a 16 h treatment by 100 µg/mL MPs or NPs; verapamil (50 µM) and FTC (10 µM) were additionally used as reference inhibitors of P-gp and BCRP, respectively. Data are the means ± SD of 3 independent assays. **, p**<0.01 and ***, p**<0.001 when compared to control cells not exposed to plastic particles or transporter inhibitors.
<figure><img src="image_7.png" /><figcaption>Fig. 2. Effects of MPs/NPs on (A) TEER and (B) Lucifer Yellow passage across Caco-2 cell monolayers. Caco-2 cell monolayers were exposed to 100 µg/mL MPs or NPs for 16 h before determination of (A) TEER values or (B) Lucifer Yellow passage. Data are the means ± SD of 11 independent experiments. ***, p<0.001 and ****, p<0.0001 when compared to control Caco-2 cells not exposed to MPs or NPs. The condition “no cells” refers to culture inserts without cells.</figcaption></figure>

were first exposed to MPs or NPs for 16 h, corresponding to an incubation time in the range of those previously used for studying interactions of PS particles with intestinal cells (Wu et al., 2019; Zeng et al., 2024). Apical or basolateral compartments of Caco-2 cell cultures as donor compartments were next incubated with 10 µM rhodamine 123 (a reference P-gp substrate) or 3.86 nM [³H]-E3S (a reference BCRP substrate), in the absence or presence of transporter reference inhibitors (50 µM verapamil or 10 µM fumitremorgin C for P-gp or BCRP inhibition, respectively). These substrate incubations were performed in HBSS medium adjusted to pH = 6.5 (apical compartment) or to pH = 7.4 (basolateral compartment). After a 90 min incubation at 37°C, receiving compartments (basolateral when the donor compartment is apical and apical if the donor compartment is basolateral) were collected and rhodamine 123 content was determined by spectofluorimetry ($\lambda_{excitation}$=485 nm, $\lambda_{emission}$=535 nm), whereas that of [³H]-ES3 was measured by scintillation counting. The apparent permeability ($P_{app}$) of these compounds was next determined using the following Eq. (1):

$$P_{\text{app}} = dQ/dt \times 1/(A \times C_D) \quad (1)$$

with $dQ/dt$ = variation of over time substrate concentration, A = cell growth area of the transwell and $C_D$ = initial concentration of substrate in donor compartment.

Apical to basolateral permeability ($P_{app}$A-B) and basolateral to apical permeability ($P_{app}$B-A) were finally considered to calculate the efflux ratio according to the Eq. (2):

$$\text{Efflux ratio} = P_{\text{app}} B-A / P_{\text{app}} A-B \quad (2)$$

## 2.9. RNA isolation and analysis

Total RNAs were isolated from HepaRG cells or Caco-2 cells using the TRIzol reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were next performed as already reported (Le Vee et al., 2013). The sequences of gene primers are indicated in Table S3. Relative quantification of the steady-state target mRNA levels was performed after normalization to the 18 S rRNA endogenous reference, through the 2($-\Delta C_t$) method. Data were finally expressed as arbitrary units, knowing that the amount of 18 S rRNA was arbitrarily set to $10^6$ arbitrary units (Moreau et al., 2011), or as percentages of

mRNA expression found in control cells not exposed to plastic particles.

## 2.10. Data analysis

Data are usually expressed as means ± SD of at least three independent assays. The analysis of variance (ANOVA) and Dunnett's post-hoc test were performed using the Prism 8.3 software (GraphPad, San Diego, CA, USA). The significance criterion was $p < 0.05$.

# 3. Results

## 3.1. Characterization of PS particle size

PS MPs and NPs suspended in water (100 µg/mL) exhibited a size average of 5.2 µm or 51.3 nm (Table S4), respectively, which agrees with specifications of the particle suppliers. When suspended in transport assay medium, in HepaRG cell culture medium or in Caco-2 cell culture medium, the size of NPs increased to 155.3 nm, 223.4 nm or 111.7 nm (Table S4), demonstrating some aggregation of NPs in these media. Such changes in NP sizes according to the medium have been previously described (Domenech et al., 2021; Lin et al., 2021; Xu et al., 2021) and may be due to putative NP aggregates or to potential adsorption of proteins and/or nutrients from the transport assay or culture media on the surface of PS particles, as already discussed (Yan et al., 2023). The size of MPs in transport assay or culture media was unfortunately not characterized, due to the fact that the used analytical method, i.e., Mastersizer S™-based light diffraction analysis, requires a dilution of samples in pure water and therefore does not permit a direct measurement in culture media; some increased size of MPs in culture media may however be expected, as for NPs and as previously described (Saenen et al., 2023).

## 3.2. Effects of PS particles on ABC transporter activity

As shown in Fig. 1A, MPs and NPs, no matter the concentration used here, failed to significantly increase accumulation of rhodamine 123 in P-gp-positive MCF7R cells and neither did they impair or increase CDCF or Hoechst 33342 retention in MRP-positive HuH-7 cells and HEK-BCRP cells, respectively. PS particles thus failed to directly inhibit ABC transporter activities. By contrast, ABC transporter inhibitors cyclosporin A, probenecid and fumitremorgin C significantly increased cellular accumulation or retention of the ABC transporter substrates
<figure><img src="image_8.png" /><figcaption>Fig. 4. Effects of MPs/NPs on cellular viability of Caco-2 cells and HepaRG cells. Caco-2 cells and HepaRG cells were exposed to 1, 10 or 100 µg/mL MPs or NPs for 48 h before determination of cell viability using the MTT assay. Data are expressed as percentages of cellular viability of control cells not exposed to MPs/NPs, set at 100 %, and are the means ± SD of at least 3 independent assays. Dashed lines indicate viability of control cells.</figcaption></figure>

failed to decrease accumulation of fluorescent substrates for SLCs
handling organic cations (OCT1 and OCT2) or organic anions
(OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3) in HEK-293 trans-
porter-overexpressing cell clones. These SLCs are physiologically found
at the sinusoidal pole of hepatocytes (OCT1 and OATPs) or at the
basolateral pole of proximal tubular cells (OCT2 and OATs) (Giacomini
et al., 2010). This most likely indicates that PS MPs and NPs failed to
inhibit SLC transporter activities. By contrast, reference blockers of SLC
activities, i.e., amitriptyline (inhibiting OCTs), probenecid (inhibiting
OATs), bromosulfophthalein (inhibiting OATP1B1), rifamycin SV
(inhibiting OATP1B3) and erlotinib (inhibiting OATP2B1) significantly
reduced accumulation of SLC substrates (Fig. 3), demonstrating the
considered SLC transporters were fully functional in SLC-overexpressing
HEK-293 cells.

3.4. Effects of PS particles on ABC and SLC transporter mRNA expression

Effects of PS particles on human drug transporter expressions were
investigated in Caco-2 cells and HepaRG cells, which have been previ-
ously shown to represent relevant cellular models when studying drug
transporter levels and regulations (Bucher et al., 2014; Martin et al.,
2008). Caco-2 cells constitutively expressed MRP2, MRP3 and OATP2B1
at a high level (mRNA expression ≥ 20 arbitrary units), MDR1/P-gp and
BCRP at moderately level (5 arbitrary units ≤ mRNA expression < 20
arbitrary units) and OCT1 at a low level (mRNA expression < 5 arbitrary
units) (Fig. S1). HepaRG cells exhibited high levels of MRP2, MRP3,
OATP2B1 and OCT1 expression, unlike MDR1/P-gp which was moder-
ately expressed and BCRP found to be weakly expressed (Fig. S1), in
agreement with previous findings (Le Vee et al., 2013). Treatments by 1,
10 or 100 µg/mL PS NPs or MPs for 48 h did not decrease the viability of
Caco-2 cells or HepaRG cells (Fig. 4) and did not alter their morphology
(data not shown). Exposure to Caco-2 cells to MPs or NPs failed to
significantly impair mRNA expression of MDR1/P-gp, MRP2, MRP3,
BCRP, OATP2B1 and OCT1 (Fig. 5A). Similarly, treatment of HepaRG
cells by MPs or NPs did not hinder mRNA expression of these trans-
porters (Fig. 5B).

4. Discussion

Plastic particles attract growing interest owing to their wide distri-
bution in the environment, considering they can be airborne or found in

drinking water and food. This probably results in a wide exposure of
humans to these pollutants (Borriello et al., 2023), which may trigger
toxic effects (Zarus et al., 2021). The exact threat they may represent
towards human health remains yet poorly characterized (Lim, 2021;
Wardman et al., 2021). In this context, the analysis of possible in-
teractions of plastic particles with human cellular and molecular pro-
cesses is worthy of interest, allowing to identify or exclude human
proteins as possible targets for plastic particles. The data reported in the
present study indicate that human ABC and SLC drug transporters,
already known to interact with environmental contaminants, including
plastic-associated chemicals (Tastet et al., 2023a), are not regulated
with respect to their activity or mRNA expression by exposure to PS MPs
or NPs used at concentrations up to 100 µg/mL. These human drug
transporters are therefore unlikely to be direct and univocal targets for
synthetic PS particles. They may be consequently presumed to not play
any role in the passage/translocation of MPs or NPs across physiological
barriers, reported notably at the intestinal, brain and placental level
(Medley et al., 2023; Meng et al., 2023; Yang et al., 2022). However, a
protective role of ABC transporters against reactive oxygen species
induced by nanomaterials cannot be excluded (Yuan et al., 2021).

The lack of effects of PS MPs and NPs, used at concentrations of up to
100 µg/mL in the present study, towards the activities of P-gp, MRPs and
BCRP is coherent with the lack of modulation of calcein acetomethoxyl
ester (AM) accumulation in human gastric AGS cells in response to a
24 h treatment by 128 µg/mL PS NPs (Lin et al., 2021). Calcein AM is a
substrate for P-gp (Holló et al., 1994) and is rapidly hydrolyzed by
cytoplasmic esterases into fluorescent calcein, a substrate for MRPs
(Feller et al., 1995). Activities of the ABC transporters P-gp and MRPs
are therefore not impaired by NPs in gastric AGS cells, as well as in the
P-gp and/or MRP-positive cells used in the present study, i.e., P-gp-ex-
pressing mammary MCF7R cells, hepatic MRPs-positive HuH-7 cells and
P-gp-expressing Caco-2 cells. By contrast, calcein accumulation has been
shown to be increased in Caco-2 cells exposed to PS NPs (20–80 µg/mL)
and MPs (80 µg/mL) for 12 h (Wu et al., 2019) or in human liver THLE-2
cells exposed to 0.2 µg/mL NPs for 90 days (Chen et al., 2023), sug-
gesting that PS particles can block ABC transporter activities in such
cells. The reason for such discrepancies with respect to inhibition of ABC
transporters by plastic particles remain unclear. They could reflect dif-
ferences in cell exposure to NPs/MPs, such as the presence or the
absence of FCS or the acute or chronic nature of the treatment by the
particles. In this context, it is noteworthy that exposures to NPs/MPs in
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Effects of MPs/NPs on human drug transporter mRNA expression in (A) human intestinal Caco-2 cells and (B) human hepatic HepaRG cells. Caco-2 and HepaRG cells were exposed for 48 h to MPs or NPs used at 1, 10 or 100 µg/mL. Transporter mRNA expression was then determined by RT-qPCR. Data are expressed as percentages of transporter expression found in control cells not exposed to MPs/NPs, arbitrarily set at 100%; they are the means ± SD of 4 independent assays. Dashed lines indicate transporter mRNA levels in control cells.</figcaption></figure>
the present study were performed in the presence of FCS, in order to mimic *in vivo* physiological scenarios, where NPs/MPs are in contact with human proteins and nutrients, notably at plasma level. Moreover, times of cellular exposure to NPs/MPs in the present study (ranging from 5 min to 16 h for activity assays) are in the range of those usually retained when judging the direct interactions of chemicals with transporters (Brouwer et al., 2013; Tastet et al., 2023b), whereas longer treatment time, such as a 90-day exposure (Chen et al., 2023), may primarily lead to phenotypic alterations of cells, leading secondarily to indirect alterations of transporters. In this context, NPs/MPs-mediated impairments of membrane barriers, including the intestinal barrier (Liang et al., 2021; Zhang et al., 2023), may reflect alterations of structural components of these barriers, such as tight-junction impairment (L. Li et al., 2023; Yang et al., 2021) or membrane lipid destabilization (Fleury and Baulin, 2021), rather than direct targeting of transporters. In the same way, inhibition of ABC transporter activity by graphene and graphene oxide nanomaterials or by multiwalled carbon nanotubes is probably due to alterations of plasma membrane structure and fluidity, and not to direct interactions of these carbon nanomaterials with transporters (Liu et al., 2016; Yu et al., 2016). Finally, it is noteworthy that the NPs/MPs-related increase of calcein accumulation in Caco-2 cells reported previously by Wu et al. is rather low, i.e., by approximately 20% (Wu et al., 2019), which indicates that NPs/MPs may in fact decrease very moderately ABC transporter activity in Caco-2 cells. Such data and our present results may therefore be recapitulated in the unifying conclusion that PS MPs/NPs do not or only weakly inhibit ABC transporter activities, thus excluding any major effects.

Additionally, PS NPs and MPs failed to alter mRNA expression of ABC or SLC drug transporters in intestinal Caco-2 cells and hepatic HepaRG cells, thus supporting the global lack of interactions of these PS particles with drug transporters. In comparison, polymethylmethacrylate (PMMA) and methyl formaldehyde resin (MF) particles have been recently shown to reduce MRP2 mRNA levels in HepaRG cells and to induce BCRP mRNA expression in both HepaRG cells and Caco-2 cells (Paul et al., 2023). These effects may reflect specific effects of these PMMA and MF particles when compared to PS particles. This supports the idea of differential behaviors of PMMA, MF and PS particles towards cells, already reported for uptake and transport processes (Paul et al., 2022), and highlights the need to consider NPs and MPs of different materials/polymers when addressing global plastic particle toxicity issues.

PS MPs and NPs, used up to 100 µg/mL for 48 h, failed to impact cell morphology and viability of Caco-2 cells and HepaRG cells, thus discarding any major toxic effects of these plastic particles towards these cells. The lack of impairment of TEER and Lucifer Yellow passage across Caco-2 cell monolayers also supports this conclusion. A similar lack of PS particle cytotoxicity has been reported in various *in vitro* studies (Domenech et al., 2020; Jing et al., 2023; Zhang et al., 2022), even if PS particles may in fact trigger some cellular responses such as reactive oxygen species production or inflammatory processes, without concomitantly inducing major toxic effects (da Silva Brito et al., 2023; Schirinzi et al., 2017; Zeng et al., 2024). Nevertheless, other studies described alteration of cell viability in PS particles-treated cells (Guo et al., 2023; Hu et al., 2022; Koner et al., 2023; Z. Li et al., 2023), linked notably to mitochondrial injury (Y. Li et al., 2023). These rather discordant results may be due to the nature of the plastic particles used in the assays, including their chemical component, their size and their concentration. The nature of the cells treated by plastic particles and the experimental conditions of the assays, such as the exposure time, are also key parameters to consider, as discussed above. A standardization of *in vitro* assays aimed at characterizing plastic particle toxicity, with exclusion of biases and artifacts (Petersen et al., 2022), would be therefore worthy of interest. The consideration of low doses of NPs/MPs, relevant for environmental exposure, has to be additionally prioritized (Lenz et al., 2016), as well as that of true-life (and not prepared and of commercial origin) plastic particles (Rajendran and Chandrasekaran,

2023), even if the plastic particle doses to which humans are exposed remain debated (Pletz, 2022; Senathirajah et al., 2021).

## 5. Conclusion

Synthetic PS MPs and NPs used at various concentrations, including one environmentally-relevant (1 µg/mL), did not inhibit *in vitro* activity of human ABC and SLC drug transporters. They also did not regulate their expression in human intestinal or hepatic cells. Taken together, these data support the conclusion that drug transporters do not constitute direct and univocal major targets for PS particles. It is however noteworthy that plastic particles usually carry a wide range of chemicals, including plastic additives and also adsorbed environmental pollutants (Abafe et al., 2023; Zimmermann et al., 2021), which are for some of them, established inhibitors and/or substrates for drug transporters (Nicklisch and Hamdoun, 2020; Tastet et al., 2023a). Whether the association/adsorption of these chemicals on plastic particles may impact their interactions with drug transporters would deserve further studies. Besides, the present study focused on PS particles, used for rather short-term treatments (up to 48 h). Additional studies are therefore required to investigate (i) the possible interactions between drug transporter proteins and other major types of MPs and NPs present in the environment, including polyethylene terephthalate (PET), polyvinyl chloride (PVC) or polyethylene (PE) particles, and (ii) the potential long-term effects of chronic exposure to plastic particles on drug transporter expression and activity.

### CRediT authorship contribution statement

**Valentin Tastet:** Writing – original draft, Validation, Investigation, Formal analysis, Conceptualization. **Olivier Fardel:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Nolwenn Brandhonneur:** Methodology. **Arnaud Bruyere:** Validation, Supervision, Methodology, Conceptualization. **Marc Le Vee:** Validation, Supervision, Methodology, Conceptualization. **Alexis Verger:** Methodology.

### 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.

### Data Availability

Data will be made available on request.

### Acknowledgments

This study has received funding from the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) under Grant Agreement N° 2020/01/124. and from the European Union's Horizon Europe research and innovation program under Grant Agreement N° 101057014 (European Partnership for the Assessment of Risks from Chemicals). Valentin Tastet is the recipient of a fellowship from the Région Bretagne. The authors would like to thank Mrs Sylvie Rohou for her expert technical assistance.

### Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.etap.2024.104563.
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