# In vitro assessment of aryl hydrocarbon, estrogen, and androgen receptor-mediated activities of secondary organic aerosols formed from the oxidation of polycyclic aromatic hydrocarbons and furans

Abd El Rahman El Mais, Barbara d'Anna, Alexandre Albinet, Selim Aït-Aïssa

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## To cite this version:

Abd El Rahman El Mais, Barbara d'Anna, Alexandre Albinet, Selim Aït-Aïssa. In vitro assessment of aryl hydrocarbon, estrogen, and androgen receptor-mediated activities of secondary organic aerosols formed from the oxidation of polycyclic aromatic hydrocarbons and furans. Environmental Research, 2025, 273, pp.121220. 10.1016/j.envres.2025.121220. ineris-04991387

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# *In vitro* assessment of aryl hydrocarbon, estrogen, and androgen receptor-mediated activities of secondary organic aerosols formed from the oxidation of polycyclic aromatic hydrocarbons and furans

Abd El Rahman El Mais<sup>a,b,2 ID</sup>, Barbara D'Anna<sup>b</sup>, Alexandre Albinet<sup>a,*,1 ID</sup>, Selim Aït-Aïssa<sup>a,**,1</sup>

## ARTICLE INFO

**Keywords:**

Biomass burning

Secondary organic aerosols (SOAs)

Oxidation flow reactor (OFR)

*In vitro* cell-based bioassays

Aryl hydrocarbon receptor (AhR)

Toxicity

## ABSTRACT

Biomass burning constitutes a significant source of fine particulate matter (PM<sub>2.5</sub>) in the atmosphere, particularly during winter due to residential wood heating. This source also emits substantial quantities of volatile and semi-volatile organic compounds, leading through (photo-)chemical and physical processes, to the formation of secondary organic aerosols (SOAs), accounting for a significant fraction of PM<sub>2.5</sub>. The current understanding of the biological effects of SOA resulting from the oxidation of major gaseous precursors emitted by biomass burning (e. g., polycyclic aromatic hydrocarbons (PAHs), phenols, furans) is still limited. Mechanism-based *in vitro* cellular bioassays targeting toxicologically relevant modes of action have proven valuable in assessing and quantifying the overall biological activity of complex mixtures like SOA, thereby revealing the presence of toxicologically relevant compounds. The main objective of this study was to investigate, using a battery of *in vitro* mechanism-based cellular bioassays, the aryl hydrocarbon (AhR), estrogen (ER), and androgen receptor (AR)-mediated activities of laboratory-generated SOA resulting from the oxidation of four PAHs and three furans. SOA was produced using an oxidation flow reactor (OFR) under either daytime (OH radicals) or nighttime (NO<sub>3</sub> radicals) conditions. Furan-derived SOA did not exhibit any biological activity with the targeted endpoints. PAH-derived SOA, formed from AhR weakly or inactive PAHs, showed significant AhR-mediated activities. Notably, SOA resulting from naphthalene and acenaphthylene + acenaphthene demonstrated the highest AhR activation potency, with greater activities observed for SOA formed through NO<sub>3</sub> radical oxidation. No endocrine-disrupting activity was observed for the PAH-derived SOA, similar to the individual parent PAHs (with the exception of fluorene and phenanthrene PAHs which were weekly anti-androgenic). These findings underscore the substantial contribution of PAH-derived SOA to the AhR-mediated activities of PM.

## 1. Introduction

Particulate matter (PM) exposure is associated with adverse health effects such as cancer, respiratory and cardiovascular diseases, neurological disorders, and increased mortality (Brook et al., 2010; Kim et al., 2015; Pope et al., 2002, 2020; Rajagopalan et al., 2018; Thurston et al., 2016). Residential wood combustion (RWC) releases large amounts of PM (Chen et al., 2022; Denier van der Gon et al., 2015; Olsen et al., 2020; Srivastava et al., 2018; Viana et al., 2016; Vicente and Alves, 2018; Weber et al., 2019; Zhang et al., 2019) and volatile and

semi-volatile organic compounds (VOCs and SVOCs) (Ahern et al., 2019; Akherati et al., 2020; Bruns et al., 2016; Hartikainen et al., 2018; Hatch et al., 2015, 2017, 2018; Rúžičková et al., 2022). VOCs and SVOCs can undergo (photo-)chemical and physical processes leading to the formation of secondary organic aerosols (SOAs) (Hallquist et al., 2009; Kroll and Seinfeld, 2008; Ziemann and Atkinson, 2012), accounting significantly for the total organic aerosols and fine particle (PM<sub>2.5</sub>) concentrations in ambient air (Ciarelli et al., 2017; Jiang et al., 2019; Kelly et al., 2018; Olsen et al., 2020).

Polycyclic aromatic hydrocarbons (PAHs) and furans emitted by

<footnote><sup>a</sup> Institut National de l'Environnement Industriel et des Risques (INERIS), Parc Technologique Alata, Verneuil-en-Halatte, 60550, France</footnote>

<footnote><sup>b</sup> Aix Marseille Univ, CNRS, LCE, Marseille, France</footnote>

<footnote>* Corresponding author.</footnote>

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

<footnote>E-mail addresses: alexandre.albinet@ineris.fr (A. Albinet), selim.ait-aissa@ineris.fr (S. Aït-Aïssa).</footnote>

<footnote n="1">¹ Equal contribution.</footnote>

<footnote n="2">² Current affiliation: Institut National de l'Environnement Industriel et des Risques (INERIS), Parc Technologique Alata, Verneuil-en-Halatte, 60550, France.</footnote>
RWC are important SOA precursors (Ahern et al., 2019; Akherati et al., 2020; Bruns et al., 2016; Rúžičková et al., 2022; Tiitta et al., 2016; Yee et al., 2013). Aromatic compounds (including monoaromatics, PAHs, and phenols) and furans account for about 10–50% (with PAHs contributing 10–20%), and 5–40%, respectively of the total VOCs emitted by biomass burning (Bhattu et al., 2019; Bruns et al., 2016; Hatch et al., 2015; Schauer et al., 2001). Through homogeneous (in gaseous phase) or heterogeneous (gas/particle) reaction processes with atmospheric oxidants such as ozone ($O_3$), hydroxyl (OH) or nitrate ($NO_3$) radicals, PAHs form oxygenated and nitrated derivatives (oxy- and nitro-PAHs) associated to SOA (Atkinson and Arey, 2007; Kautzman et al., 2010; Keyte et al., 2013; Ringuet et al., 2012). These compounds are also emitted concomitantly with PAHs by RWC (Nalin et al., 2016; Olsen et al., 2020; Shen et al., 2012; Vicente and Alves, 2018). While the atmospheric reactivity of PAHs and furans is well documented (Al Ali et al., 2022; Aschmann et al., 2011, 2014; Bierbach et al., 1995; Jiang et al., 2020; Keyte et al., 2013; Kind et al., 1996; Li et al., 2018; Newland et al., 2022; Ringuet et al., 2012; Zheng et al., 2006), the level of scientific understanding in terms of SOA formation and biological activities is still limited.

PAHs, their oxy and nitro derivatives, and furans are known to cause serious risks to human health due to their toxicity and carcinogenicity (Clergé et al., 2019; Environmental Health Criteria (EHC) 229, 2003; IARC, 2013, 2012, 2010; Idowu et al., 2019; Kim et al., 2013; Kirkok et al., 2020; Peng et al., 2023). One major mode of action of these species is their ability to interact with intracellular receptors such as the aryl hydrocarbon receptor (AhR) (Machala et al., 2001; Misaki et al., 2007; Peng et al., 2023; Vogel et al., 2020; Vondráček et al., 2017). AhR is a well-known xenobiotic sensor sensitive to various environmental organic chemicals, including dioxins, planar polychlorinated biphenyls, polycyclic aromatic compounds, and certain pesticides. Upon binding to such xenobiotics, activated AhR modulates the transcription of various genes involved in several physiological processes such as xenobiotic metabolism, immunity, or development. Among them, cytochrome P450 1A (CYP1A) plays a central role in xenobiotic metabolism, leading either to xenobiotic elimination or the bio-activation of certain pro-carcinogens (e.g., PAHs) into reactive metabolites, potentially leading to DNA damage and carcinogenesis (Hahn, 2002; Hahn et al., 2009; Stockinger et al., 2014; Vogel et al., 2020). Beyond AhR activation, PAHs and their derivatives can also modulate the activity of some endocrine-related nuclear receptors, such as the estrogen (ER) and androgen receptors (AR), either through direct receptor binding (Marin-Kuan et al., 2017; Misaki et al., 2023; Pěnčíková et al., 2019; Vinggaard et al., 2008) or indirectly via cross-talk with activated AhR (Ohtake et al., 2003, 2007). Both ER and AR are transcription factors that regulate the expression of key genes involved in the regulation of the endocrine system. The dysregulation of their normal signaling can have physiological consequences, including immunosuppression, carcinogenesis, (neuro)developmental disorders, and reproductive defects (De Coster and van Larebeke, 2012; Janošek et al., 2006).

SOAs are complex mixtures containing thousands of different chemical species, many of whose identities and associated toxicities remain largely unknown. Identifying the key toxic constituents represents a major challenge in human health risk assessment. Although target chemical analytical methods are sensitive and capable of detecting trace levels, they cannot describe and quantify all the individual constituents of SOA or identify unknown bioactive compounds. As a complementary approach, bio-analytical tools, such as *in vitro* mechanism-based cellular bioassays, serve as more integrative tools. These high-throughput bioassays allow for quantifying the overall biological activity by focusing on biologically active molecules, including those not yet identified, and accounting for potential mixture effects (Brack et al., 2016; Eichbaum et al., 2014; Escher et al., 2020).

Several studies have implemented this approach and demonstrated that ambient PM can elicit AhR-mediated, ER-mediated (estrogenic and/or anti-estrogenic), and AR-mediated (androgenic and/or anti-

androgenic) effects (Klein et al., 2006; Novák et al., 2009, 2013, 2014, 2020; Nováková et al., 2020; Wenger et al., 2009; Zhang et al., 2018). To our knowledge, no study has investigated the AhR-, AR-, and ER-mediated activities of SOA.

In this work, a battery of *in vitro* mechanism-based bioassays targeting several endpoints i.e., PAH- and dioxin-like activities (AhR-mediated), (anti)estrogenicity (ER and AER), and (anti)androgenicity (AR and AAR), was implemented to investigate and compare the biological responses of SOA formed from different precursors (PAHs and furans) and through different oxidation processes (OH vs. $NO_3$ radicals).

## 2. Materials and methods

### 2.1. Laboratory generation of SOA

The SOA generation procedure has been previously described in detail (El Mais et al., 2023). Briefly, SOA was generated, at ambient temperatures ($T = 24–32 °C$) and environmentally relevant relative humidity ($RH = 25–62%$), using a potential aerosol mass-oxidation flow reactor (PAM-OFR, Aerodyne Research) (Kang et al., 2007; Lambe et al., 2011) by the reaction between OH or $NO_3$ radicals with individual furan or PAH SOA precursors (Fig. A1). OH radicals were produced by photolysis of $O_2$ using four UV lamps emitting at $λ = 185$ and 254 nm (OFR185 method) (Li et al., 2015; Peng and Jimenez, 2020). The photolysis of $O_2$ at 185 nm produces $O_3$ which is photolyzed at 254 nm to produce $O^{(1)D}$. $O^{(1)D}$ then reacts with water vapor (introduced using a Nafion membrane humidifier, Perma Pure LLC, FC100-80-6-MKK) to generate OH radicals. In order to maximize the SOA generation, the lamps' voltage was adjusted, depending on the precursor studied, from 1.8 to 3 V. The corresponding irradiance (Ir), continuously measured by a photodiode integrated inside the PAM-OFR (Tocon_C6, sglux), ranged from 6 to 71 µW cm$^{-2}$ (Table A1). The OH exposure ($OH_{exp}$), was evaluated experimentally by measuring the degradation of $SO_2$ injected into the PAM-OFR during additional experiments (El Mais et al., 2023) and also using the KinSim chemical kinetic solver (Peng and Jimenez, 2019). $OH_{exp}$ ranged from $1.05 × 10^{10}$ to $2.57 × 10^{12}$ molecule cm$^{-3}$ s, corresponding to about 0.1–20 equivalent aging days (Table A1), assuming a daily average OH radical concentrations of $1.5 × 10^6$ molecules cm$^{-3}$ (Finlayson-Pitts and Pitts Jr, 2000; Mao et al., 2009). $NO_3$ radicals were generated from the thermal decomposition of $N_2O_5$ at room temperature in a dark OFR (OFR-i$N_2O_5$ method) (Lambe et al., 2020). The gaseous $NO_3$ and $N_2O_5$ were produced via the reaction of two distinct reagent flows of 100 ppm of $NO_2$ (from a cylinder of 1% $NO_2$ in $N_2$, Air Liquide) and 200 ppm of $O_3$ ($NO_2:O_3$ ratio of 0.5) in a perfluoroalkoxy (PFA) laminar flow reactor (LFR, 152.4 cm long × 2.22 cm ID) coupled to the PAM-OFR. The $NO_3$ exposure ($NO_{3exp}$), evaluated using the KinSim model, ranged from $1.68 × 10^{12}$ to $2.10 × 10^{14}$ molecule cm$^{-3}$ s, corresponding to about 0.05–5.7 equivalent nights of 14 h (Table A2) considering an average nighttime $NO_3$ mixing ratio of $7.36 × 10^8$ molecules cm$^{-3}$ (Asaf et al., 2010).

Three furans [furan (99% purity), 2-methylfuran (2-MF, 99%) and 2,5-dimethylfuran (2,5-DMF, 99%)], and four PAHs [naphthalene (Naph, 99%), acenaphthylene (Acy, 75%), the remaining 25% are acenaphthene (Ace)), phenanthrene (Phe, 99%), and fluorene (Flu, 98%)] have been selected and investigated. They were all purchased from Sigma-Aldrich (France) and introduced into the PAM-OFR in their pure state, without $NO_x$ or seed particles. Furans and PAHs were injected at relatively high concentrations ranging from 9 to 50 mg m$^{-3}$ (3–13 ppmv) and 0.2–15 mg m$^{-3}$ (0.03–3 ppmv), respectively (Tables A1 and A2) to produce stable (up to 17 h, Fig. A2) SOA concentrations (ranging from 0.05 to 3 mg m$^{-3}$) and collect enough PM quantities (Table A3) for *in vitro* bio-assessment. SOA generated has been also monitored using a set of online instrumentation, described in Appendix A (Section A.I), to get insights into their physicochemical properties (El Mais et al., 2023).
## 2.2. Filters sampling and determination of the PM mass and their carbonaceous content

SOA has been collected using a constant flow sampler (6.5 L min⁻¹; QB1, DADOLAB) on quartz fiber filters (Pallflex Tissuquartz, Ø = 47 mm) without PM size cut. Before sampling, filters were calcined at 500 °C for 12 h to remove any organic contamination. At the end of each experiment, lasting between 4 and 17 h, one filter was collected. Three to six replicates were performed for each SOA precursor and each oxidant (n = 54 in total). After sampling, filters were placed in individual Pall Analyslide Petri dishes, sealed in polyethylene bags, and stored at -20 °C until extraction. Given the stable SOA generation, the mass of SOA collected for each experiment was determined from the Q-ACSM (Quadrupole Aerosol Chemical Speciation Monitor; Aerodyne research) measurements (Fig. A2). SOA mass concentrations were considered equivalent to organic matter (OM) for OH radical experiments, and equal to OM + NO₃ (particulate nitrate) for NO₃ radical experiments. The collected PM quantities ranged from 0.2 to 16 mg (Table A3). Finally, PM carbonaceous content, elemental carbon-organic carbon (EC-OC), and total carbon (TC) have been determined on 1 cm² quartz fiber filter punches, by a thermo-optical method using a Sunset Lab analyzer and following the EUSAAR-2 protocol (Cavalli et al., 2010).

## 2.3. Filters extraction and sample preparation

Depending on the sample loading (based on PM mass and TC), a full filter, a filter section, or two filters have been extracted. Extraction was performed using a quick easy cheap effective rugged and safe (QuECh-ERS)-like procedure allowing to efficiently extract the organic species adsorbed on PM while maintaining the sample integrity (Albinet et al., 2013, 2014, 2019). Filters were placed in glass centrifuge tubes with 7 mL (or 10 mL for two filters) of acetonitrile (ACN; VWR, HPLC gradient grade) and then vortexed for 3 min at 1500 rpm (Digital Multi Tube Vortexer, DVX-2500, VWR) (Fig. A3). After agitation, samples were centrifuged at 4500 rpm for 5 min (Sigma, 3–16 PK centrifuge). The supernatant (approximately 5.5 mL) was collected and filtered through a 0.2 µm Uptidisc PTFE. For furan SOA samples with low PM loadings, up to 4 filter extracts were pooled. Sample extracts were then divided into two fractions by weighting (balance precision = 0.0001 g). The first half was collected in 10 mL vials and stored at -20 °C for other studies, while the second was concentrated under a gentle nitrogen stream to near dryness and resuspended in ACN (200 or 250 µL). 100 µL was kept as a backup and the remaining volume (100 or 150 µL) was exchanged to dimethyl sulfoxide (DMSO, Sigma Aldrich) and then kept at -20 °C until in vitro bio-analyses.

## 2.4. In vitro bioassays

The in vitro AhR-, ER- and AR-mediated activities of filter extracts were evaluated using the human cell lines HepG2 (ATCC#HB-8065), MELN (Balaguer et al., 1999), and MDA-kb2 (Wilson et al., 2002), respectively. Procedures for routine cell culture are described in detail in Appendix A (section A.III). Procedures for exposure to reference chemicals or sample extracts were previously described in detail (Aït-Aïssa

et al., 2010; Louiz et al., 2008; Serra et al., 2020). In brief, cell exposure was conducted in 96-well culture microplates. HepG2, MELN, and MDA-kb2 cells were seeded at an optimum density of approximately 5 × 10⁴, 8 × 10⁴, and 6 × 10⁴ cells per well, respectively (Table 1). After 24 h, the cells were exposed to serial dilutions of sample extracts, solutions of SOA precursors, or reference chemicals, all initially prepared in DMSO and then diluted in a culture medium.

Exposure was performed in triplicates within the same plate. Each microplate included a negative control (DMSO), and a positive control, namely 10 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for PAH- and dioxin-like AhR-mediated activities, 10 nM of 17β-estradiol (E2) for ER activities, and 10 nM of dihydrotestosterone (DHT) for AR activities. For antagonism assays, co-exposures with agonists at a concentration leading to 60–80% of maximal response (i.e., E2 at 60 pM or DHT at 0.3 nM) were carried out to investigate anti-estrogenic (AER) or anti-androgenic (AAR) activities, respectively. The final DMSO concentration was constant and did not exceed 0.5% and 0.6% (v/v) for agonism and antagonism assays, respectively. These solvent concentrations have no toxic effect and do not modify the cell response. MELN and MDA-kb2 cells were exposed for 24 h, while HepG2 cells were exposed for both 4 and 24 h, to distinguish between transient PAH-like activity mediated by readily metabolized compounds after 4 h, such as PAHs, and dioxin-like activity mediated by more persistent compounds (24 h), such as dioxins (Louiz et al., 2008). The biological responses of reference molecules [TCDD, benzo[a]pyrene (B[a]P), E2, hydroxytamoxifen (OH-Tam), DHT, and flutamide (Flut) for dioxin-like, PAH-like, estrogenicity, anti-estrogenicity, androgenicity, and anti-androgenicity, respectively] were assessed in parallel to each assay. In addition, the biological activities of the SOA precursors (PAHs or furans) were also measured for concentrations ranging from 0.03 to 50 µM.

At the end of exposure, the luciferase activity based on luminescence detection was measured in MELN and MDA-kb2 cell lines. The culture medium was removed by plate inversion and replaced by 50 µL per well of medium containing 471 µM of luciferin (Xenolight D-luciferin potassium salt, 30 mg mL⁻¹). Luminescence was then measured in living cells after 5 min. Ethoxyresorufine-O-deethylase (EROD) activity was measured in exposed HepG2 cells. After emptying the plates, 100 µL of culture medium containing 5 µM of 7-ethoxyresorufin (7-ERF, Sigma) was added to each well, and the plates were left to incubate for 10 min at 37 °C before stopping the reaction by adding 100 µL per well of DMSO. Fluorescence was measured at 530 nm (excitation) and 590 nm (emission). Both luminescence and fluorescence signals were measured with a BioTek Synergy H4 Hybrid Microplate Reader. The results were expressed as a relative response to the positive control (in %) specific to each cell line included in each plate.

In parallel to AER and AAR assays, the cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). Following luminescence measurements, the plate was emptied and 100 µL per well of medium containing 0.5 mg mL⁻¹ of MTT was added. Cells were incubated for 3 h allowing the reduction of MTT by metabolically active cells into a blue formazan precipitate. After incubation, the MTT-containing medium was removed and replaced by 100 µL per well of DMSO. The plates were shaken for 10 min, and absorbance was measured at 570 nm against a 640 nm reference wavelength. The results were expressed as percentages relative to

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

List of the *in vitro* bioassays used to assess the AhR, ER, and AR-mediated activities of the laboratory-generated SOA.</caption><thead><tr><th>Endpoint/Receptor</th><th>Human cell line, tissue</th><th>Seeding density (cell/well)</th><th>Microplate type</th><th>Reference chemical</th><th>Detection method</th></tr></thead><tbody><tr><td>AhR 4 h: PAH-like</td><td>HepG2, liver</td><td>5 × 10<sup>4</sup></td><td>Transparent</td><td>B[a]P<br>TCDD</td><td>Fluorescence (EROD)</td></tr><tr><td>AhR 24 h: Dioxin-like</td><td></td><td></td><td></td><td>E2</td><td>Luminescence (Luc)</td></tr><tr><td>Estrogenicity (ER)</td><td>MELN, breast</td><td>6 × 10<sup>4</sup></td><td>White opaque</td><td>OH-Tam</td><td></td></tr><tr><td>Anti-estrogenicity (AER)</td><td></td><td></td><td></td><td>DHT</td><td>Luminescence (Luc)</td></tr><tr><td>Androgenicity (AR)</td><td>MDA-kb2, breast</td><td>8 × 10<sup>4</sup></td><td>White opaque</td><td>Flut</td><td></td></tr><tr><td>Anti-androgenicity (AAR)</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
DMSO-exposed control cells.

## 2.5. Data treatment and Bio-TEQ calculations

Dose-response curves of SOA precursors, reference chemicals, and sample extracts were modeled according to the Hill equation model using the RegTox 7.0.7 Microsoft Excel™ macro (Vindimian et al., 1983). To ensure comparability across samples within a given bioassay, the Hill maximal effect parameter was fixed at 100%, corresponding to the maximal effect of the reference compound. Modeled curves were then used to derive EC<sub>20</sub> and IC<sub>20</sub>, effective concentration inducing or inhibiting 20% of the effect, respectively.

The biological equivalent concentrations of the sample extracts (Bio-TEQ<sub>extract</sub>) were determined using Eq. (1) as the ratio of the EC<sub>20</sub> or IC<sub>20</sub> of the Ref to that of the sample extract. Bio-TEQ<sub>extract</sub> were expressed as reference compound equivalents (Ref eq.) per L of culture medium.

$$ \text{Bio-TEQ}_{\text{extract}} = \frac{\text{EC}_{20\text{Ref}}}{\text{EC}_{20\text{extract}}} \quad \text{or} \quad \frac{\text{IC}_{20\text{Ref}}}{\text{IC}_{20\text{extract}}} \qquad (1) $$

Bio-TEQ concentrations were then expressed for 1 µg of PM using the PM mass collected on the filter and extracted (see section 2.2).

## 2.6. Quality assurance/Quality control (QA/QC)

### 2.6.1. Background contamination, particle loss measurements, and experimental blanks collection

The PAM-OFR was kept clean by systematically flushing with clean air (ambient air + total filter + activated charcoal scrubber) and keeping the lamps at the maximum voltage (10 V, Irradiance (Ir) ≈ 200 µW cm<sup>-2</sup>) when switching from one experiment to another or during the weekend. Furthermore, the particle number was checked before all experiments, at the exit of the PAM-OFR, with lamps at 1.8 V. If it was below 100 particles cm<sup>-3</sup>, the system was considered not contaminated. In addition, measurements of the particle transmission through the PAM-OFR, in the range of 30–200 nm, were carried out using ammonium sulfate aerosols (obtained by nebulizing 0.5 or 1 mM of (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> aqueous solutions using an atomizer coupled to diffusion dryer (TSI, 3076 and 3062) to correct for any potential particle losses. Results showed particle loss (in number and mass) below 5% for all PM sizes, therefore no particle wall-loss correction was applied. Finally, before starting the generation and collection of SOA from a given SOA precursor, experimental blanks were collected for about 1-2 h (n = 22 for both OH and NO<sub>3</sub> radicals). To do this, oxidants were generated under the same conditions as for the SOA generation, but without injecting PAH or furan SOA precursors into the PAM-OFR.

### 2.6.2. Laboratory and experimental blanks biological assessment and data treatment

For each extraction batch, laboratory blanks (solvent and calcinated filter blanks, n = 4), were performed to check for any potential contamination occurring during the sample extraction procedure. All laboratory blanks showed no response to the studied endpoints. The tested experimental blanks (n = 16) did not induce any activity for all endpoints except for the PAH-like one where a very weak activity was observed (Fig. 5, A5, A6, and A7). This response depended on the number of filters/extracts pooled. As specified earlier, for low PM sample loadings several filters/extracts were pooled (up to 4 filters) to increase the sample extract concentrations. To ensure that the procedure did not introduce any bias into the sample bioanalysis, the exact protocol was repeated using experimental blank filters. It was observed that when a single blank filter was extracted, the resulting response was consistently negative. However, combining multiple experimental blank filters occasionally led to the detection of weak PAH-like activity. In such cases, the experimental blank activity was quantified as Bio-TEQ<sub>extract</sub> and compared to the Bio-TEQ value of the corresponding samples. If the Bio-TEQ value of a sample was three times lower than the Bio-TEQ<sub>extract</sub> was classified as inactive.

### 2.6.3. Inter-assay variability based on reference chemicals

The inter-assay consistency of the cell response and the sensitivity of the assays were evaluated using the response of the reference ligands throughout the whole study. The measurements were conducted over 5 months, using the same batch of cells specific to each assay. The EC<sub>20</sub> or IC<sub>20</sub> values for each reference molecule, as shown in Fig. 1, were consistent over the whole period and fell within the same order of magnitude. The EC<sub>20</sub> (mean ± SD, n = 4–13) were about 95 ± 38 pM for TCDD (dioxin-like, AhR 24 h), 10 ± 4 nM for B[a]P (PAH-like, AhR 4 h), 130 ± 65 pM for DHT (AR), and 14 ± 3 pM for E2 (ER). For antagonism assays, IC<sub>20</sub> was equivalent to 194 ± 74 nM for Flut (AAR) and 1.5 ± 0.5 nM for OH-Tam (AER). These average values were in agreement with the known sensitivity of the bioassays (Aït-Aïssa et al., 2010; Balaguer et al., 1999; Serra et al., 2019). They were then used to calculate the Bio-TEQs (Eq. (1)). The variabilities observed, retrieved from the coefficient of variation (CV), ranged from 22% for ER to about 40% for PAH-like, dioxin-like, AR, and AER and 50% for AAR assays. This inter-assay variability, which was in the upper range as compared to the values reported in other studies for similar bioassays (Garoche et al., 2023; Keiter et al., 2009; Kunz et al., 2017), is considered as good to acceptable as it integrates biological response within a long-term use of the same batch of cells.

### 2.6.4. Evaluation of the method reproducibility based on the analysis of the SRM extracts

The overall reproducibility of the method was evaluated using extracts of a known quantity (44–96 mg) of the standard reference material SRM 1649b (urban dust) (NIST, 2016), which was included in each extraction batch (n = 10). SRM samples were extracted and processed identically to SOA samples. The Bio-TEQ concentrations for SRM 1649b were reported for a reference mass of 1 g based on the extracted dry mass. The Bio-TEQ results were corrected for moisture content, which was determined as 4.29% on average through four independent measurements using an HR 73 moisture analyzer (Mettler Toledo).

<figure><img src="image_5.png" /><figcaption>Fig. 1. Independent determination of EC<sub>20</sub> or IC<sub>20</sub> of the reference molecules for the different in vitro assays over the whole study. The circles represent the independent determinations (n = 4–13).</figcaption></figure>
# 3. Results and discussion

## 3.1. Biological profiles of SRM 1649b

Fig. 2 and Fig. A4 show the biological responses induced by SRM 1649b. This reference material exhibited significant AhR-mediated PAH-like and dioxin-like activities, as well as anti-androgenic and anti-estrogenic activities. No detectable estrogenic or androgenic activities were observed. The Bio-TEQ values (mean ± SD, n = 3–10) were about 6 ± 2 $mg_{B[a]P}$ eq. g$^{-1}$, 0.7 ± 0.4 $µg_{TCDD}$ eq. g$^{-1}$, 1 ± 0.5 $mg_{Flut}$ eq. g$^{-1}$, and 4 ± 2 $µg_{OH-Tam}$ eq. g$^{-1}$ for PAH-like, dioxin-like, AAR, and AER activities, respectively. The variability observed ranged from 39% for PAH-like activity to approximately 50% for dioxin-like, AAR, and AER activities (Fig. 2). Chemical analyses of PAHs and PAH derivatives performed on the same SRM extracts demonstrated variability of less than 10%, indicating that the primary source of variability was likely due to cellular response behavior rather than chemical inconsistencies.

The obtained *in vitro* activity profiles aligned with the chemical composition of SRM 1649b, which is known to contain many AhR active chemicals such as PAHs, oxy- and nitro-PAHs, dioxins, furans, poly-chlorinated biphenyls (PCBs), halogenated pesticides, phenolic species, etc. (Albinet et al., 2019, 2014, 2013, 2006; Andrysik et al., 2011; NIST, 2016; Wise et al., 2006, 2000). To our knowledge, this study is the first to report the ability of this reference material to elicit AER and AAR activities, expanding its relevance for toxicological profiling.

## 3.2. *In vitro* activities of SOA samples

### 3.2.1. AhR-mediated activity

Fig. 3 illustrates the PAH-like activities (AhR, 4 h exposure) induced by PAH and furan SOA formed via day- and nighttime oxidation processes. Furan SOA generated through both oxidation pathways exhibited no detectable activity, and their SOA precursors, tested up to 10 µM, were also inactive (Fig. A5). In contrast, PAH SOA remarkably demonstrated significant PAH-like activities (Fig. 3), while the parent PAHs were found inactive (Naph, Flu, Phe) or weakly active (Ace + Acy, EC₂₀ of 1.24 µM) in this assay (Fig. A5), which aligns with the anticipated lack or minimal AhR activity of these PAHs, as reported in previous studies (Louiz et al., 2008; Machala et al., 2001; Murahashi et al., 2007). Interestingly, the PAH-like activity of PAH SOA varied depending on the SOA precursor and the oxidant involved. For daytime oxidation, all PAH SOA exhibited PAH-like activities, with Naph SOA showing the highest activity, achieving an average Bio-TEQ of 20 ± 11 $ng_{B[a]P}$ eq. $µg_{PM}^{-1}$. Acy + Ace, Flu, and Phe SOA displayed comparable activities, approximately 19 times lower than those of Naph SOA. PAH-like activities increased

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** *In vitro* PAH-like (AhR 4 h), dioxin-like (AhR 24 h), anti-androgenic (AAR), and anti-estrogenic (AER) activities of NIST SRM 1649b (urban dust). Results are expressed as reference equivalent (Ref eq.) per gram of SRM on a dry-mass basis. The circles represent each independent determination (n = 3–10).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** PAH-like biological responses (AhR, 4 h exposure, $ng_{B[a]P}$ eq. $µg_{PM}^{-1}$) for PAH and furan SOA formed via day- and nighttime oxidation processes. Missing boxplots indicate inactive samples. Furan and fluorene (Flu) SOA generated with $NO_3$ radicals are not shown due to unstable SOA generation or the absence of SOA formation. The circles represent each independent determination (n = 1–4).</figcaption></figure>

substantially (by factors of 3–12) for PAH SOA formed with $NO_3$ radicals, highlighting the critical role of nocturnal chemistry when addressing the toxicity of SOA. Naph SOA again exhibited the highest activity, with an average Bio-TEQ of 234 ± 119 $ng_{B[a]P}$ eq. $µg_{PM}^{-1}$, followed by Acy + Ace SOA (6 ± 1 $ng_{B[a]P}$ eq. $µg_{PM}^{-1}$), and finally Phe SOA (3 $ng_{B[a]P}$ eq. $µg_{PM}^{-1}$).

The dioxin-like activities (AhR, 24 h exposure) induced by PAH and furan SOA formed during both day- and nighttime chemical processes are shown in Fig. 4 and Fig. A5. Overall, the dioxin-like activity patterns parallel those of PAH-like activity, suggesting that detected activities are due to the same compounds. Persistence after 24 h may be due either to different concentrations and/or to different potency to bind to and activate AhR. Similar to the PAH-like activities, neither furans nor their corresponding SOA exhibited any dioxin-like activity. Among the PAH SOAs, only those derived from Naph and Acy + Ace, formed via reactions with OH or $NO_3$ radicals, were biologically active. As for PAH-like activity assay, Naph SOA demonstrated the highest potency for both reactivity processes, with Bio-TEQ levels 7 and 18 times higher than those of Acy + Ace SOA for OH and $NO_3$ radicals, respectively. A marked increase in biological responses was observed from OH to $NO_3$ reactivity, with Naph SOA increasing 10-fold (from 9 ± 3 to 86 ± 5

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Dioxin-like activities (AhR, 24 h exposure, $p_{TCDD}$ eq. $µg_{PM}^{-1}$) for PAH and furans SOA formed via day- and nighttime oxidation processes. Missing boxplots indicate inactive samples. Furan and fluorene (Flu) SOA generated with $NO_3$ radicals are not shown due to unstable SOA generation or the absence of SOA formation. The circles represent independent determinations (n = 1–4).</figcaption></figure>
$pg_{TCDD}$ eq. $\mu g_{PM}^{-1}$) and Acy + Ace SOA increasing 4-fold (from 1.3 to 5 ± 2 $pg_{TCDD}$ eq. $\mu g_{PM}^{-1}$). The lack of dioxin-like activity for Phe and Flu SOA suggested that AhR-active compounds in these SOAs are rapidly metabolized by cells after 24 h of exposure, consistent with previous findings (Louiz et al., 2008). Overall, nighttime chemical processes significantly amplified AhR-mediated biological responses, with Naph and Acy + Ace SOA emerging as the most potent.

To our knowledge, this study is the first one to identify AhR-
mediated activity of PAH SOA using such a bioassay-based approach.
It clearly demonstrates that bioactive PAH-like compounds are gener-
ated from AhR-inactive parental PAHs, while their chemical identity is
yet unknown. Hydroxy-PAHs, polyaromatic ketones, and quinones are
well-known compounds formed, in gaseous and particulate phases, from
the oxidation of PAHs in the presence of O$_3$, OH, and NO$_3$ radicals
(Kautzman et al., 2010; Keyte et al., 2013; Lannuque and Sartelet, 2024;
Perraudin et al., 2007; Riva et al., 2016, 2017; Sasaki et al., 1997; Zhou
and Wenger, 2013a, 2013b). These compounds are known to interact
with the AhR receptor, triggering its mediated effects (Bekki et al., 2009;
Misaki et al., 2007; Peng et al., 2023; Vogel et al., 2020) and are likely
responsible for the AhR-mediated activities observed in this study. Thus,
the identification of the specific chemical species responsible for these
biological activities is a crucial step toward understanding the mecha-
nisms underlying the observed effects.

3.2.2. Endocrine activities

Fig. 5, A6, and A7 present the ability of PAHs and furans and their corresponding SOA to activate (agonistic assay) or block (antagonistic assay) the estrogen and androgen receptors. For AR-mediated activities (Fig. A6), no androgenic activity was observed with the SOA precursor compounds or their corresponding SOA formed with OH or NO3 radicals. This aligns with previous studies reporting the lack of androgenicity in PAHs and their derivatives (Croes et al., 2016; Weiss et al., 2009). In the antagonism assays (Fig. 5), a progressive decline in signal was observed for 2-MF SOA formed with OH radicals and Naph SOA with both OH and NO3 radicals, suggesting potential antagonistic effects on the AR. However, parallel MTT tests revealed that this decrease was primarily due to cytotoxicity observed at the same concentrations. Consequently, no AR or AAR activity was conclusively evidenced in the generated SOA.

Among the SOA precursor compounds, only Flu and Phe, exhibited weak anti-androgenic activity with IC20 values of 9.2 ± 2.8 µM and 5.0 ± 0.3 µM, respectively.

Similarly, for the ER-mediated activities (Fig. A7), none of the SOA precursor compounds or their corresponding SOA elicited an activation or inhibition of the estrogen receptor. As with the AR assay, the decline in signal observed for 2-MF and Naph SOA was predominantly linked to cytotoxic effects rather than anti-estrogenic activity. In summary, the generated SOA did not induce any detectable ER, AR, AAR, or AER activities.

4. Conclusion

This study represents the first evaluation, using a suite of
mechanism-based cellular bioassays, of the biological activities of SOA
formed from various SOA precursors typically emitted by biomass
burning and under different oxidation conditions (day- and nighttime
processes). The findings revealed no detectable AhR or endocrine-
disrupting activities for individual furans (furan, 2-MF, and 2,5-DMF)
or their corresponding SOA, regardless of whether they were formed
via OH or NO3 radical oxidation. No endocrine activity was observed for
the PAH-derived SOA as for the individual PAHs. In contrast, PAH SOA
exhibited significant AhR-mediated activities, notably a PAH-like
pattern, while the PAH SOA precursors were weakly (Acy) or entirely
inactive (Naph, Flu, Phe). This highlights the critical role of atmospheric
aging in the formation of bioactive species that can interact with and
alter the normal functioning of nuclear receptors, potentially leading to
adverse health effects. Among the tested SOAs, Naph and Acy + Ace SOA
demonstrated the highest potency, with AhR-mediated activity sub-
stantially amplified under nocturnal oxidation conditions involving NO3
radicals. This underscores the heightened significance of nighttime
chemical processes in determining the toxicological profile of SOA.

Future work should focus on identifying the chemical species
responsible for these biological activities. This challenging but essential
task will provide a deeper understanding of the observed effects and aid
in assessing the risks associated with SOA. Employing integrated ap-
proaches, such as effect-directed analysis, that combines sample frac-
tionation, effect-based assays and advanced chemical characterization,

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** MDA-kb2 anti-androgenicity assay responses to the reference ligand (Flutamide, in red), individual PAHs and furans (in pink), and the corresponding SOA generated through day- (blue) and nighttime (green) oxidation processes. Only fluorene (Flu) and phenanthrene (Phe) exhibited anti-androgenic effects. No samples were collected for furan and Flu SOA formed with NO3 radicals, and no assays were conducted for these cases. Bars represent mean values ± SD of four technical replicates. Results are representative of at least 3 independent determinations. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
will be invaluable for isolating, identifying, and understanding the
bioactive compounds within SOA mixtures (Brack et al., 2016; Creusot
et al., 2013).

CRediT authorship contribution statement

Abd El Rahman El Mais: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. *Barbara D'Anna:* Writing – review & editing, Supervision, Investigation, Formal analysis. *Alexandre Albi-net:* Writing – review & editing, Validation, Methodology, Supervision, Resources, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization. *Selim Aït-Aïssa:* Writing – review & editing, Validation, Supervision, Resources, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

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

Acknowledgments

This work has been supported by the French Ministry of the
Environment.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envres.2025.121220.

Data availability

Data will be made available on request.

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