Inhalation toxicity of arsenic-containing mine dust in an air-liquid interface bronchial epithelial model.

Background
Occupational exposure to airborne particles remains a critical public health concern across many industrial sectors, with mining industries occupying a prominent position due to frequent exposure of workers to high concentrations of respirable dust [1]. Prolonged exposure to mineral particles triggers a complex series of biological alterations within the respiratory system [2, 3]. From a pathophysiological perspective, numerous molecular and microenvironmental responses contribute to persistent inflammation, prolonged tissue injury, and structural remodeling. Over time, these processes frequently result in the progression of interstitial fibrosis and the onset of lung cancer [4–6]. Presently, a considerable number of miners are actively engaged in operations at the Gejiu tin mine in Yunnan, where they are subjected to prolonged exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons (PAHs), and radioactive radon, thereby significantly elevating the risk of lung cancer [7, 8]. A comprehensive etiological study of lung cancer among Gejiu miners has demonstrated that the pathogenesis of lung cancer is associated with the synergistic interactions between metallic elements and non-metallic components present in tin mine dust (MD) (such as radon and its progeny, arsenic, lead, and iron) [9–11]. Despite these epidemiological insights, all of which are based on studies from 20 years ago, a significant gap remains in our understanding of the mechanistic underpinnings that translate environmental exposures into cellular pathology. Understanding the adverse health effects caused by mineral particles remains limited at present, primarily due to the lack of in vitro test systems that can accurately mimic human exposure to MD particles and elucidate the underlying mechanisms in biological cells. Therefore, in order to explore the toxicity differences among various tin MD, we designed in vitro simulation experiments to evaluate the inhalation toxicity and specific toxic effects of different MD.
Traditionally, in vitro toxicological assessments have relied on submerged cell culture models in which the test substance is diluted in a liquid medium [12]. However, these conditions inadequately mimic the in vivo scenario of aerosol deposition on respiratory epithelium under realistic conditions. Although such models have offered useful insights into dose-response relationships and mechanistic endpoints, they inherently lack the physiological relevance of the lung environment. This discrepancy limits the translational potential of findings obtained under submerged conditions and highlights the need for more realistic exposure systems [13]. Air-liquid interface (ALI) culture models have emerged as a superior alternative, as they recapitulate the unique microenvironment of the respiratory epithelium and allow for controlled exposure to aerosolized substances in a manner that closely mimics in vivo inhalation [14]. In this context, the air-liquid interface cell exposure system (ALICE) represents a cutting-edge technological advancement that emerged at the right moment, facilitating the precise and reproducible deposition of aerosolized particles directly onto cultured lung cells and thereby providing refined control over the cell-delivered dose [15, 16]. The integration of vibrating membrane nebulizers and combined cloud settling with single-particle sedimentation in ALICE facilitates the controlled and low-stress delivery of aerosols, enabling kinetic evaluation of dose deposition [17]. Additionally, a significant advancement accompanying ALI exposure systems is the integration of real-time dosimetry methods, such as Quartz Crystal Microbalance (QCM) [18]. This technology allows for the precise quantification of the mass of particles deposited per unit area, which is particularly important when assessing toxicants like tin MD that may have variable deposition efficiencies [19]. The ability to monitor dose deposition in real time not only enhances the reproducibility of in vitro exposures but also provides critical information, especially given the heterogeneous and dynamic nature of MD particles, which can be correlated with subsequent toxicological endpoints [20].
In the lung, the epithelial cells that line the airways and alveoli act as the primary points of contact for inhaled substances derived from environmental, occupational, or other external origins [21]. Consequently, epithelial cells serve as the main focus in cell culture models designed for inhalation toxicology studies [22]. Among these, the tumor cell line Calu-3 and the immortalized bronchial epithelial cell line 16HBE14o- have been widely utilized as representative models in vitro inhalation toxicology studies [23]. The Calu-3 cell line originates from lung adenocarcinoma, specifically from submucosal gland serous cells. Upon cultivation at the ALI for a few weeks, the resulting airway epithelial layer produces mucins, forms cilia, and exhibits the features of fully differentiated airway epithelium with a strong barrier function [24]. Previous research has demonstrated that Calu-3 cells serve as a valuable tool for screening potential chemical entities and assessing the inhalation toxicity of respirable substances [25]. 16HBE14o- cell line is an SV40-transformed human bronchial epithelial cell line [26]. It exhibits a cobblestone-like morphology, forms tightly polarized monolayers, and can be maintained in culture under ALI conditions [27]. The choice of Calu-3 and 16HBE cells in our study is based on their well-established capacity to mimic human airway epithelial responses, such as barrier function regulation, mucociliary clearance, and the secretion of proinflammatory cytokines.
In this research, we carried out a comprehensive assessment of two epithelial cell models grown under extended ALI conditions. These cell models were exposed to aerosolized tin MD using the ALICE system, and real-time deposition monitoring was accomplished by the integrated QCM. Subsequent evaluations focused on key toxicological endpoints, including cell viability, cytotoxicity, epithelial morphology, barrier function, and the expression of proinflammatory markers. Importantly, comparative analyses were performed on arsenic-containing MD versus MD lacking significant arsenic levels to determine the contribution of this heavy metal to overall toxicity. In summary, the present study aims to leverage the advantages of the ALICE system to conduct a detailed, dose-controlled in vitro analysis of the inhalation hazards associated with tin MD particles.

Material & methods

Tin MD particles preparation
We separately collected three tin ore samples from tin mines located in three distinct mining regions within Gejiu, Yunnan Province, China. The raw samples were then delivered to Noozle Fluid Technology Ltd., located in Shanghai, China, where pilot-scale ultrafine powder jet milling was employed to obtain MDs. The distribution of three tin mine particle sizes was measured by the Bettersize2600 Laser particle size analyzer (Bettersize Instruments Ltd., Dandong, China). The metal compositions of three kinds of MD particles (MD-A, MD-B, and MD-C) were analyzed by means of inductively coupled plasma mass spectrometry (ICP-MS NexION® 2000, PerkinElmer, Waltham, MA, USA), according to previously published methods [28]. Prior to SEM analysis, MD particles were dispersed in ultrapure deionized water to prepare uniform suspensions. The suspensions were homogenized through vortex mixing and probe sonication to ensure even particle distribution. Droplets from these well-dispersed suspensions were then used for SEM sample deposition. To prepare the SEM samples, a droplet of the suspension was placed onto a silicon wafer and left to dry naturally in the air for an extended period overnight. The presence of different particle size fractions was subsequently confirmed through SEM analysis. (FP2012/14 Quanta 250; FEI, Czech). Three mineral dust samples were weighed and autoclaved at 121 °C for 30 min prior to drying. Endotoxin detection was conducted using the Limulus Amebocyte Lysate (LAL) test.
The ALICE system was first characterized using aqueous 0.09% sodium chloride (NaCl) solutions as reference aerosols. Subsequently, we evaluated three distinct types of MD particles alongside silica particles (silicon dioxide, CAS 14808-60-7, with 80% of the particle sizes ranging between 1 and 5 μm and a purity of 99%). Particle suspensions preparation: Four separate suspensions were prepared for comparative analysis, with each undergoing thorough homogenization through two complete cycles of vortex mixing (1 min each) followed by probe sonication (1 min each), incorporating brief pauses between cycles to prevent thermal degradation of samples. Stock suspensions were freshly prepared at three concentrations (1, 5, and 10 mg/mL) by suspending precisely weighed quantities of particles (5, 25, and 50 mg, respectively) in 5 mL of ultrapure deionized water, with all suspensions prepared immediately before nebulization to ensure sample integrity. A Qsonica Q700 sonicator was used, equipped with a titanium alloy tip probe of 3 mm in diameter. Sonication was performed for 8 min total time in an ice-water bath using a pulse cycle of 5 s ON and 2 s OFF at 40% amplitude. Following sonication in water, the particle suspension was diluted with the required volume of 0.09% NaCl solution to achieve the final concentration for nebulization. This mixture was then vortexed for 2–3 min immediately before loading into the nebulizer to ensure uniformity.
The specific remarks for particle aerosolization are as follows. (1) Prior to each aerosol exposure run, 200 µL ultrapure water containing 1% isotonic NaCl solution was nebulized for removal of residual particles from the nebulizer. The output rate of the nebulizer was measured after each nebulization and should not exceed 10% deviation from the original output rate (Table S1). (2) At the beginning of a working day, the deposited dose of 200 µL of ultrapure water with 1% (v/v) isotonic NaCl solution (blank value) was measured. (3) QCM cleaning was performed between nebulizations. The entire QCM crystal was wiped carefully with water on a Q-tip, along with the edges and every surface that came in contact with nebulized material very carefully. The procedure was repeated with 70% ethanol. The device was allowed to dry for at least 5 min to ensure complete drying of the crystal, as reflected by a stable QCM signal. (4) Four particle suspensions were prepared in water with the desired concentration (Table S2). For each particle, three technical replicates were performed. (5) For the four types of particle suspensions, a cleaning process was performed after each discharge to prevent membrane pore blockage and maintain output efficiency. Cleaning was also conducted when switching between materials. (6) The output rate of the nebulizer was measured after each nebulization. If the time exceeds 10% of the starting value, the nebulizer was placed in a beaker with ultrapure water or 70% ethanol and sonicated in an ultrasonic bath for 5–10 min. Then, the output rate was measured again. If nebulizing time did not improve, the nebulizer was cleaned more thoroughly or replaced. (7) The particle size distribution of the mine dusts and silica tested in this study indicated that all particles were smaller than 5 μm. Additionally, we provide scanning electron microscopy (SEM) images that illustrate the particle size measurements on the membrane of the Transwell insert following nebulization (Fig. S1). Furthermore, a comparison of the traditional nebulization method versus the optimized method used in this study, highlighting the improved particle distribution and reduced clogging, is presented in Fig. S2.

Cell culture
Human bronchial epithelial cells 16HBE14o- and Calu-3 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). They were cultured at 37 °C in a humidified atmosphere containing 5% CO2 (Sanyo MCO-18AIC incubator). Calu-3 cells (passages 5–12) were cultured in Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin solution.16HBE14o- cells (passages 5–12), the culture medium was Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 1% penicillin-streptomycin solution. All reagents for cell culture were purchased from Gibco Life Technologies (Gibco, Grand Island, NY, USA). Both cell lines were seeded in 25 cm2 tissue culture flasks (Falcon, New York, USA). Once the cells reached 90% confluency, they were enzymatically dissociated using 0.05% trypsin-EDTA. The cells were then seeded on the apical side of 12-well Transwell inserts (0.4 μm pore polyester membrane, 0.9 cm2 effective growth area; Corning, New York, USA) at a density of 2.0 × 105 cells/ cm2. The medium volumes in the apical and basal compartments of the insert were 0.5 mL and 1.0 mL, respectively. To prevent overgrowth prior to ALI culture, the cells were maintained under submerged conditions for 2–3 days to form a confluent monolayer. Subsequently, the cells were then air-lifted by removing the apical medium and replacing the basal medium, thereby maintaining medium-cell contact without exerting hydrodynamic pressure on the cell layer. Following a 24-hour acclimatization period at the ALI, both 16HBE and Calu-3 cells demonstrated the formation of tight junctions (TJs) and secretion of a thin surfactant layer on the apical surface.

The air-liquid interface cell exposure systems (ALICE)
All aerosol exposure experiments utilized a VITROCELL®Cloud12 device (VITROCELL® Systems, Waldkirch, Germany), which was fitted with an Aeroneb® Lab nebulizer (with a pore size of 4–6 μm, output rate of 1.39 mL/min, Aerogen, Ireland) and a VITROCELL® Cloud 12 Exposure chamber. The technical specifics regarding the principle and configuration of the QCM, as well as the ALICE. A schematic illustration of the cell exposure conditions is presented in the Supplementary Video. The ALICE base was preheated to 37 °C prior to use, with the cover (exposure top) remaining in place. Simultaneously, 250 µL of the particle suspension or water (as a blank) was mixed with 2.5 µL of an isotonic sodium chloride solution (spiked with 0.09 mg/ml NaCl to provide sufficient ions for proper operation of the vibrating mesh nebulizer). Mass deposition onto the cells was quantified using a quartz crystal microbalance (QCM 200/25, Stanford Research Systems, Sunnyvale, CA, USA), which was positioned on the ground plate of the exposure chamber. The cells grown at the ALI were taken out of the incubator, the medium in the lower chamber was changed to 1 mL of serum-free culture medium, and then the inserts were moved to the exposure chamber of the ALICE system. The QCM data acquisition was initiated, and the system stability at the zero point was monitored for 1 min. Thereafter, the cells were exposed to aerosol droplets containing MD particles generated from nebulizing a 200 µL liquid suspension. To achieve the targeted dose range, the optimal nebulization time was set to 15–60 s, corresponding to a flow rate of 0.2–0.8 mL/min. Subsequently, after an additional waiting period of 6 min (bringing the total elapsed time to 7 min), the chamber cover (exposure top) was opened to allow the deposited sample on the QCM to dry for 1 min under ambient conditions. The cover was then replaced on the ALICE system to prevent potential artifacts in the QCM signal caused by ventilation and temperature fluctuations (refer to the results section for further details). Data acquisition was stopped after an additional 3 min, and the mean value of the last 30 s of QCM data was recorded as the QCM readout representing the cell-delivered dose. After exposure, the inserts were incubated for 24 h, 48 h, and 72 h, respectively, and subsequently analyzed for cell viability (CCK8 assay) and cytotoxicity (LDH assay) as toxicological endpoints (see below). Three independent biological replicates were conducted to ensure the reliability of the results. A representative visualization of nebulizing 200 µL of ultrapure water with 1% (v/v) isotonic NaCl solution is shown in the Supplementary Video.

Cell viability assay (CCK8)
Cell viability of 16HBE and Calu-3 was determined using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China) following the manufacturer’s instructions. After exposure, the cultured 16HBE and Calu-3 epithelium at the insert were incubated for 24 h, 48 h, and 72 h, respectively, and directly treated with the test solutions. The stock solutions were prepared in propylene glycol (PG) diluted in serum-free medium to the final concentrations. For the CCK-8 assay, following flavoring treatments, the culture medium was removed from exposed cells on culture inserts and washed twice with phosphate-buffered saline (PBS). The CCK-8 reagent was mixed with serum-free cell culture medium, added to the apical side of the insert (1:10 dilution), and then incubated with 5% CO2 at 37 °C for 2–4 h. Then the culture medium was shaken well and transferred to a 96-well cell culture plate (with 100 µL/well). Afterwards, the mixture was measured by a microplate reader at 450 nm wavelength (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA, USA). There was a blank well without cells, with the wells containing only CCK-8 reagent, and the wells containing CCK-8 reagent, medium, and cells in the culture plate were used as control wells. Both the experimental wells and the blank well were exposed to MDs. The OD value of the samples was calculated, and cell viability (%) was calculated as (OD Experimental Wells - OD Blank Wells) / (OD Control Wells - OD Blank Wells) ×100%.

Cytotoxicity assay (LDH)
The LDH cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany) was used to measure the release of the intracellular enzyme lactate dehydrogenase (LDH), which is an indicator for cell membrane perforation. The test was performed on basal medium in triplicate. In each of the 5 wells of the 96-well plate, 30 µL of basal medium was mixed with 70 µL of fresh DMEM cell medium, followed by the addition of 100 µL of dye solution to all wells. The plate was then wrapped in aluminum foil and gently shaken at 20 RPM for 15 min. At the end, 50 µl HCl was added to the wells to stop the reaction. The absorbance at 492 nm was subsequently measured using a microplate spectrophotometer (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA, USA). The absorbance measurement was performed in triplicate. The concentration of released LDH was then derived from a standard curve that covers a suitable concentration range. The maximum possible LDH level (positive control) was obtained from lysing the negative control cells (unexposed) with the Lysis buffer and subsequent detection of LDH release. The results were normalized to the positive control and the negative control. (LDH in the basal compartment of the unexposed incubator control)

Cell proliferation assay (EdU)
Cells were seeded on the apical side of the insert and cultured under ALI conditions for 24 h, 48 h, and 72 h, respectively. EdU cell proliferation staining was performed using an EdU kit (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488, Beyotime, China). Briefly, 5-Ethynyl-20-deoxyuridine agent was added to each insert and allowed to incorporate for 2 h. The cells were fixed in 4% PFA for 15 min and washed three times with PBS containing 3% bovine serum albumin (BSA). The cells were incubated in PBS containing 0.3% TritonX-100 for 15 min, followed by BSA-containing PBS washes three times. The cells were stained with 5 µg/mL Hoechst 33,342 at room temperature for 10 min, washed three times with PBS, and mounted using antifade mounting medium. Images were acquired using an inverted fluorescence microscope (THUNDER Imager 3D Live Cell, Leica Microsystems, Wetzlar, Germany). Images were compiled using ImageJ, and control wells were done concurrently.

Measurement of transepithelial electrical resistance
An essential physiological function of epithelia is to provide barriers that regulate the movement of water and solutes into and out of the body. Consequently, an effective barrier function is a critical characteristic for any epithelial model. Transepithelial electrical resistance (TEER) was measured using an EVOM 2 Epithelial Voltohmmeter and STX2 electrodes (World Precision Instruments, Inc., Sarasota, FL, USA) by applying a ± 20 µA square wave alternating current across the monolayers at 12.5 Hz [31–33]. Different culture durations were employed, with 1 mL of medium in the outer well and 500 µL of medium in the Transwell insert. The insert was incubated for 30 min to ensure stabilization. Subsequently, the medium was refreshed, and TEER was measured to evaluate the integrity of the epithelial layer. The resistance of cell-free Transwell inserts was measured for control purposes and proved to be minimal (< 1 Ω·cm2). The measured TEER values were corrected by subtracting the mean resistance of blank porous membranes. The corrected measurement value was multiplied by 0.9 (the surface area of the Transwell membrane in cm2) to obtain the TEER value (Ω·cm2).

Cellular RNA extraction and quantitative real-time PCR
Total RNA was extracted from 16HBE and Calu-3 cells using TRIzol reagent according to the manufacturer’s instructions (#DP419, Tiangen, Beijing, China). Immediately following the post-incubation period, the cells were washed with PBS and lysed directly on the insert membrane by adding 350 µl of a cell lysis buffer that is suitable for total RNA isolation. First-strand cDNA was generated from 2 µg of total RNA using oligo-dT to prime the reverse transcription reaction, according to the manufacturer’s protocol. (#KR116, FP205, Tiangen, Beijing, China) The PCR primers were purchased from Shanghai Bioengineering Ltd. (Shanghai, China). Real-time PCR was performed using a Real-Time PCR System 7500 (Applied Biosystems, Carlsbad, CA, USA). The PCR conditions were as follows: 15 min of denaturation at 95 °C, followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s. All the samples were run in triplicate. The target mRNA levels were quantified with real-time qPCR using fluorogenic probe/primer combinations specific for IL-1β, IL-6, IL-8, TNF-α, CCL2, mucus protein mucin 5AC (MUC5AC), and GAPDH. The relative gene expression levels among each group were quantified using the 2−△△Ct method (relative). The primers for RT-qPCR are listed in Supplementary Table S3.

Immunofluorescence
16HBE and Calu-3 cells were analyzed by immunofluorescence staining to visualize the expression and localization of zona occludens-1 (ZO-1), E-cadherin, and MUC5AC. The ALI cells were fixed with 4% paraformaldehyde for 30 min at room temperature, washed with phosphate-buffered saline (PBS) three times, and then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Non-specific binding sites were blocked with 5% (v/v) bovine serum albumin (BSA) in PBS for 2 h at room temperature and then incubated overnight at 4 °C with diluted primary antibodies. Rabbit polyclonal anti-ZO-1 (#13663; Cell Signaling Technology, Boston, MA, USA) and E-cadherin (#3195; Cell Signaling Technology, Boston, MA, USA) and mouse monoclonal anti-MUC5AC (#61193; Cell Signaling Technology, Boston, MA, USA) antibodies were used. Next, the cells were washed with PBS three times and incubated for 1 h at room temperature with a goat anti-rabbit or anti-mouse IgG secondary antibody conjugated to Alexa 488 or 555 (Cell Signaling Technology, Boston, MA, USA) diluted 1:2000 in blocking buffer. The transwell membrane was excised and then washed in PBS for 30 min. The cells were mounted with antifade mounting medium with DAPI (#P0131, Beyotime, Shanghai, China). Images were acquired using an inverted fluorescence microscope (THUNDER Imager 3D Live Cell, Leica Microsystems, Wetzlar, Germany).

Statistical analysis
All data were expressed as mean ± standard deviation (SD) from three independent experiments performed in triplicate. The statistical analysis was performed using Graphpad Prism 10 (GraphPad Software Inc., San Diego, CA). A one-way analysis of variance (ANOVA) with a subsequent Tukey’s Multiple Comparison test was performed. Cells exposed to water (Vehicle) at the ALI served as a control for aerosol exposures. For CCK8 and LDH assays, values from cells exposed to MDs or control and from cells kept in the incubator (for ALI exposure only) were included in the statistical analysis. To assess the level of proinflammatory mediators at the ALI after stimulation with MDs, a two-way ANOVA followed by a Bonferroni post-hoc test was performed to compare treated groups to controls or to compare ALI exposure. In all the analyses, p-values < 0.05 were considered significant.

Results

Characterization of MD particles
The ores extracted from the tin mines in Yunnan consist of multiple minerals, with their precise mineral compositions varying significantly from one mine to another. The three types of mineral dust utilized in this study were sourced from three distinct mining districts. As shown in Fig. 1A, the metal element content of three tin MD samples indicates that iron (Fe) accounts for more than 40% of the total metal content. (MD-A: 42.5%, MD-B: 59%, MD-C: 49.5%). Notably, MD-A was characterized by an exceptionally high arsenic (As) content, exceeding 30%. In contrast, the elemental composition of MD-B and MD-C was predominantly composed of calcium (Ca), magnesium (Mg), and aluminum (Al), exhibiting distinct geochemical profiles compared to MD-A. As illustrated in Fig. 1B, scanning electron microscopy (SEM) analysis of tin MD particles revealed that the three mechanically generated dust types, which result from ore-crushing processes during mining operations, possess irregular morphologies with fractal edges and surface asperities. The analysis using a laser particle size distribution meter showed that the particle size distribution can be characterized by the shape and span of the curve in Fig. 1C. The median aerodynamic diameter (D50), defined as the particle size at which 50% of the cumulative volume distribution occurs, was quantified for all MD samples. The particle sizes D50 of MD-A, B, and C were measured to be 2.091 ± 0.039 μm, 2.098 ± 0.023 μm, and 2.113 ± 0.012 μm, respectively. It follows that all samples were classified within the fine particulate matter range (< 2.5 μm aerodynamic diameter), and five independent experiments were conducted and repeated to ensure accurate measurement (Supplementary particle size analysis).

Experimental setup and particle concentration characterization
The experimental setup is illustrated in Fig. 2A. Nebulize 200 µL of ultrapure water with 1% (v/v) isotonic NaCl solution and check the time needed for nebulization. The output rate should not exceed 10% deviation from the original output rate of the individual nebulizer (Supplementary Table S1). Cells were exposed to three types of MD (MD-A, B, and C) as well as silica particles. In a single-exposure experiment, the vehicle control group (water) and the low-, mid-, and high-concentration groups were simultaneously exposed to the treatment using the ALICE system. Specifically, the aerosolization of particles in suspension was carried out, yielding concentrations of 1 mg/mL, 5 mg/mL and 10 mg/mL, respectively. In addition, we selected silica particles that have been studied in existing inhalation toxicity research for comparative analysis [28, 29]. The application of concentration and the measurement of deposited aerosolized dust particles are detailed in Supplementary Table S2. The rapid and controlled nebulization of MD suspensions facilitated accurate dose administration and ensured spatially homogeneous deposition onto cells cultured at the ALI. Importantly, to minimize experimental variability, cells used for each independent experiment were from the same passage number and seeding stock.
Real-time monitoring of respirable MD deposition was achieved through a quartz crystal microbalance (QCM) system [18]. As illustrated in Fig. 2B, characteristic QCM frequency responses were recorded during 200 µL aerosol deposition experiments using the ALICE system, revealing three distinct operational phases: Phase I: Aerosol Deposition Initiation (0–50 s). Upon nebulizer activation (t = 0 s), rapid aerosol cloud formation and chamber homogenization occurred, followed by progressive particle deposition on the QCM surface. The deposition rate increased linearly, reaching maximum slope (dΔf/dt) at 30 s as indicated by the steepest QCM frequency shift. System precision was quantified through 50 s stable-phase measurements (1 Hz sampling), with variation expressed as 1σ of signal stability. Phase II: Evaporation-Deposition Equilibrium (50 s–5 min). Following peak signal attainment at 60 s, competing processes dominated: aerosol deposition versus droplet evaporation. This dynamic equilibrium resulted in gradual signal attenuation until stabilization at ~ 5 min, marking the establishment of (1) a persistent liquid film on the QCM surface and (2) chamber air saturation, preventing further evaporation. Notably, the asymptotic QCM signal at this stage does NOT reflect accurate mass measurements due to residual liquid-phase interference. Phase III: Final Drying Stabilization (>5 min). Chamber decompression (top removal) induced rapid liquid film desiccation, completing solid-phase transition within seconds. This phase change manifested as an abrupt signal inflection (direction dependent on solute concentration), ultimately stabilizing at a plateau representing the true deposited mass. Critically, only this terminal asymptotic value permits valid Sauerbrey equation applications (Supplementary Eq. 1) for mass conversion from frequency shifts, as residual moisture effects are eliminated [18].

Evaluating the toxicity of MDs at the air-liquid interface
Through the precise quantification enabled by QCM, the dose-dependent cytotoxicity of MD and silica particles was systematically investigated under ALI exposure conditions. ALI cultures of 16HBE and Calu-3 cells were exposed to different concentrations of MD-A, MD-B, MD-C, and silica particles (1 µg/cm2, 5 µg/cm2, 10 µg/cm2) for 24, 48, and 72 h (Table S2). Cell viability and cytotoxicity were measured. As shown in Fig. 3A, in 16HBE cells, compared with the control group (Vehicle), MD-A showed a dose-dependent increase in cell survival rates at all time points, while MD-B and MD-C had decreased survival rates only at 24 h, with no significant changes at 48 and 72 h. Silica particles reduced cell survival after 72 h. As shown in Fig. 3B, in Calu-3 cells, MD-A significantly enhanced cell survival at all time points for high-concentration levels. MD-B reduced cell viability at 24 and 72 h, yet demonstrated an increase at 48 h. MD-C decreased viability at 24 h but increased at 48 and 72 h under high-concentration conditions. In general, Fig. 3A and B demonstrate that the cell viability of the four types of particles is correlated with dose levels rather than deposition time post-exposure. Notably, significant alterations in cell viability were observed for both 16HBE and Calu-3 cells following 24 h of exposure across different concentration levels. For both MD particles and silica, significant cytotoxicity was observed at all tested concentrations, as indicated by the release of lactate dehydrogenase (LDH). Figure 3C and D demonstrate a reduction in cytotoxicity at all time points following MD-A exposure in both cell types. Consistent with expectations, cell viability increased (Fig. 3A and B) while cytotoxicity decreased (Fig. 3C and D) across all concentration levels. Notably, we observed an increased cytotoxicity upon exposure to MD-B, MD-C, and silica in different time and concentration groups. Consequently, Calu-3 cells exhibit less pronounced cytotoxicity compared to that observed in 16HBE cells.

According to cell viability and cytotoxicity tests, MD-A was found to significantly increase cell viability and decrease cytotoxicity in two types of ALI-cultured epithelial cells (Fig. 3). To compare the effects of MD-A with those of other particulate matters on cell proliferation, we performed 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, which has been commonly used to indicate DNA synthesis, to confirm the effects of MDs on cell proliferation. The EdU assay results indicated that MD-A significantly enhanced the proliferation ability of 16HBE and Calu-3 cells compared to other particulate matters. This indicates that arsenic mineral dust possesses unique properties that promote cell proliferation. Bright-field microscopic images confirm that MD suspensions enable accurate dose delivery and spatially uniform deposition onto cells cultured at the ALI (Fig. 4).

Analysis of the barrier functions of bronchial epithelial cells
The 16HBE14o- bronchial epithelial cell line serves as a crucial cell model in airway disease research. Calu-3 cells create highly polarized monolayers featuring TJs and significant TEER values, while also exhibiting microvilli and mucin granules, as previously reported [25, 30, 31]. In our in vitro prediction analysis of cell barrier function, both the Calu-3 epithelium and 16HBE cells were utilized (Fig. S3). Upon the formation of functional TJs between cells, Calu-3 cells exhibit higher TEER values than 16HBE cells, indicating a stronger correlation with the integrity of cellular barriers. The TEER values exhibited differences among the four types of particles in Calu-3, MD-A exhibited the highest values across different doses (868.3 ± 75.1Ω·cm2, 926.6 ± 120.1Ω·cm2, 830 ± 140.5 Ω·cm2) as observed 24 h post-exposure. Exposure to MD-B and MD-C had no significant effect on TEER values. In contrast, exposure to silica led to a decrease in TEER values. (556.6 ± 75 Ω·cm2, 576 ± 102 Ω·cm2, 573.3 ± 86 Ω·cm2). 16HBE cells exhibit lower TEER values, typically less than 150 Ω·cm2; no significant variation was observed among different types of MD particles. (Fig. S4) These values fall within the reported physiological range for this cell line (120–800 Ω·cm²) [30] suggesting that the inherently low baseline TEER of 16HBE monolayers may limit the sensitivity of this assay to detect further changes under particle exposure.
Epithelial cells are structurally organized into a continuous layer and interconnected through protein-based junctions to establish a paracellular barrier. Immunofluorescence microscopy was used to visualize 16HBE and Calu-3 cells expressing the TJs protein ZO-1 and the adherens junction protein E-cadherin, as well as secreting MUC5AC. As illustrated in Fig. 5A and B, fluorescent antibody labeling shows that confluent monolayers of 16HBE and Calu-3 cells exhibit well-defined staining. After exposure to MD-A (10 µg/cm2), significant alterations were observed in the fine structure of TJ rings in Calu-3 cells compared with other particles. These changes were characterized by more tortuous cell boundaries and more obvious breaks forming in the monolayer structure (Fig. 5B(a)). Conversely, in 16HBE cells, the TJs exhibit less complex interdigitated patterns and less well-defined junctional margins compared to Calu-3 cells. (Fig. 5A(a)). In addition to MD-A, silica particles reduce the expression of ZO-1 while leaving the cell boundary structures unchanged. As shown in Fig. 5A(b) and 5B(b), the expression of E-cadherin in both cell types formed near-continuous rings and was predominantly localized to the periphery of each cell. In 16HBE, E-cadherin exhibited slight diffuse cytoplasmic localization. Silica particles resulted in a significant reduction in the expression of E-cadherin in Calu-3 and 16HBE, while other particles failed to induce any notable changes. Figure 5A(c) and 5B(c) show that MUC5AC was used to visualize the distribution of acidic mucins and revealed a punctate staining pattern characteristic of mucins. In our study, MUC5AC expression was detected in both cell types, and exposure to four distinct particulate matters significantly increased MUC5AC protein secretion, which was correlated with elevated MUC5AC mRNA expression levels.

Effects of MDs exposure on the inflammatory response
Inflammatory cytokines are one of the indicators to evaluate inflammatory reactions. As shown in Fig. 6, exposure to different mine dust particles induced variable transcriptional responses of proinflammatory cytokines and mucin genes in both 16HBE and Calu-3 cells. Among the three dust types, MD-A displayed the most distinct pattern. In 16HBE cells, MD-A elicited a significant upregulation of IL-1β, IL-6, IL-8, and MUC5AC predominantly at the high dose (10 µg/cm²), whereas TNF-α and CCL2 did not show consistent changes at this concentration. In Calu-3 cells, MD-A also elicited the strongest response at 10 µg/cm², with marked induction of TNF-α and MUC5AC and increases in other mediators compared with the vehicle control. At low and medium doses, several cytokines showed scattered or partial elevations, but the overall pattern lacked consistency. Compared with MD-A, MD-B and MD-C induced less consistent changes, often limited to isolated cytokines at specific doses without a clear dose-dependent trend. Silica served as a positive reference particle and provoked broader and more robust inflammatory gene expression across multiple mediators in both cell lines. Collectively, these results indicate that MD-A, characterized by its high arsenic content, triggers a pronounced inflammatory response only at higher doses, whereas lower doses lead to heterogeneous and less reproducible effects.

Discussion
Prolonged inhalation of airborne contaminants in occupational settings is strongly linked to a spectrum of respiratory disorders, including chronic obstructive pulmonary disease (COPD) and various forms of airway dysfunction [32]. A number of industrial exposures are well-established contributors to lung cancer risk, particularly substances such as crystalline silica, arsenic, asbestos, beryllium, and diesel exhaust. Additional carcinogenic agents include heavy metals like cadmium, nickel, and chromium [33]. Tin miners in Gejiu, Yunnan Province, China, face a significantly elevated risk of developing lung cancer due to distinct occupational exposures [34]. Qiao YL et al. [35] demonstrated that the high mortality rate of lung cancer among Yunnan tin miners is associated with occupational exposure to factors such as radon and arsenic in the production environment. Following the research by Lubin et al. [36], arsenic in the Yunnan tin MD exists as a poorly soluble compound predominantly associated with iron; the concentration of arsenic in the lungs of local miners with lung cancer is found to be 30 to 40 times higher than that in the control group. Some evidence is available suggesting that arsenic is associated with cancer [37, 38]. In a related investigation, WH Chen’s analysis of four tin mines in China suggested that the co-presence of high arsenic levels and crystalline silica in MD significantly contributes to the increased lung cancer mortality observed in these populations [39]. The synergistic effects of mixed exposures pose significant health risks, particularly in mining environments where workers are exposed to complex aerosols comprising crystalline silica, metal particulates, arsenic-containing compounds, and radon. Therefore, we independently collected MD samples from tin mines in three distinct mining regions within Gejiu, Yunnan Province, China. From their precise mineral compositions varying significantly from one mine to another, the analysis revealed that although all MD samples had high iron content (ranging from 42.5% to 59% of the total metal), there were marked differences in other elemental profiles. MD-A, characterized by an exceptionally high arsenic content (>30%), contrasts sharply with MD-B and MD-C, which were rich in calcium, magnesium, and aluminum. Such heterogeneity in elemental composition has been previously observed in mineral dust samples from various mining operations [40, 41]. In this context, the presence of elevated arsenic levels in MD-A is particularly significant, as arsenic is known to exert complex biological effects, including both cytotoxic and proliferative responses in exposed cells. The level of arsenic may be a key factor in adjusting the toxicity of particulate matter within environmental health evaluations. Upon inhalation as respirable particles, arsenic predominantly exists in the forms of inorganic arsenite (As3+) and arsenate (As5+) [42]. The toxicological characteristics of arsenic-bearing mineral dust are marked by multi-organ carcinogenicity and intricate pathophysiological interactions [43]. Understanding the full scope of arsenic’s toxicological profile is crucial for developing effective public health strategies and environmental regulations.
The ALICE system adopts system-based cloud settling together with single-particle sedimentation to serve as the mechanism for droplet deposition. This study aims to evaluate the inhalation toxicity associated with dose-controlled delivery of MD particles suspended in liquids to cell systems cultured at the ALI. Our findings provide detailed insights into the physicochemical characteristics, dose deposition, and biological responses of three distinct tin MD (MD-A, MD-B, and MD-C) as compared to well-characterized silica particles [44]. Such a comprehensive analysis not only underlines the feasibility of using ALI exposures for toxicity evaluations but also expands our understanding of how differences in particle composition can lead to different cellular outcomes. The precise characterization of MD is critical, as the health risks associated with inhalable particulate matter depend greatly on particle size, morphology, and chemical composition [45]. Therefore, studying dust containing different elements and compounds and with different proportions is meaningful. In our study, scanning electron microscopy (SEM) revealed that the dust particles possess irregular morphologies with fractal edges and surface asperities. Laser particle size distribution measurements further confirmed that the median aerodynamic diameters of these particles fall within the fine particulate matter range (< 2.5 μm). This observation is in line with previous studies that have documented the formation of fine particles during mining operations and highlighted the importance of particle size in determining the transport and deposition behavior of dust in the respiratory system [46]. In this study, which focused on inhalable particles from occupational environments, the three mineral powders with sizes below 2.5 μm were categorized as fine particles. Although their size did not reach the nanometer level, the ALICE system, widely acknowledged as a dependable nebulization platform for nanoparticles [47], was also shown to be effective in aerosolizing these particles. Deposition of MD particles using the ALICE system successfully met critical criteria for nanoparticle delivery, including precise control of exposure, dose-dependency, uniform spatial distribution, and consistent deposition efficiency across all tested dust concentrations. The findings from our study support the suitability of the ALICE system as a realistic and reliable in vitro platform for evaluating the inhalation toxicity of particulate pollutants derived from mining operations. The particle size distribution of the MDs tested primarily falls within the respirable fraction (< 5 μm), suggesting that these particles can reach the distal airways and potentially deposit in the alveolar region. However, the majority of deposition in occupational settings typically occurs in the conducting airways due to impaction and sedimentation, especially for particles in the submicron to low-micron range [48]. Therefore, the air–liquid interface (ALI) culture system using bronchial epithelial cell lines (Calu-3 and 16HBE14o-) was selected to model the upper and central conducting airway epithelium, as a primary site of initial interaction with inhaled particulates. The ALI culture system provides a structurally and functionally differentiated epithelial barrier that more accurately mimics the in vivo airway environment compared to submerged cultures. Cells grown at ALI develop apical-basal polarity, TJs, and mucociliary differentiation, all of which are critical for studying the epithelial response to inhaled particles. This system allows for direct deposition of airborne particles onto the apical surface of the cells, closely simulating the physiological route of exposure [13], and enables assessment of key endpoints such as barrier integrity (via TEER), junctional protein expression (e.g., ZO-1, E-cadherin), mucus production (e.g., MUC5AC), and inflammatory mediator release.
The hazard assessment results in this study show significant differences in toxicity between arsenic-containing and non-arsenic mineral dust. The three types of mineral dust particles studied have distinct compositions that impact their biological interactions. MD-A, which has a high arsenic concentration, exhibits opposite cytotoxicity compared to the other particles without arsenic. MD-A increased cell viability and enhanced proliferation in a dose-dependent manner in 16HBE and Calu-3 cells as measured by EdU incorporation, while the arsenic-free MD and silica decreased cell viability. This paradoxical observation, in which a particle with a high concentration of toxic metals appears to promote cell proliferation rather than induce overt cytotoxicity, highlights the potential for unique biological interactions mediated by arsenic and related compounds. Although arsenic is conventionally classified as a toxicant, low-concentration exposures have been associated with hormetic responses that may enhance cell proliferation [49]. The unique molecular structure of MD-A may interact with cellular pathways involved in growth and division, leading to enhanced cell proliferation. Nevertheless, the long-term implications of such proliferative responses remain to be elucidated, particularly in terms of potential carcinogenicity and the development of chronic respiratory diseases. Cell viability assays performed on 16HBE and Calu-3 epithelial cells demonstrated that the biological responses varied with both the concentration and the type of particulate matter. Notably, differences between the two cell lines (16HBE and Calu-3) were observed, suggesting that cell-specific factors may modulate the response to particulate matter exposure. Calu-3 cells exhibit lower sensitivity than 16HBE cells, possibly due to their culture at the ALI, forming tightly polarized monolayers with robust TJs and maintaining close opposition of adjacent cell membranes. The differential cytotoxic responses exhibited by these two cell lines, along with their unique characteristics, emphasize the crucial importance of selecting an appropriate cellular model for conducting accurate and reliable toxicological assessments. Our findings show that trace metal composition, especially arsenic content, determines the toxicological profile of MD and highlights the impact of particles’ physicochemical properties on biological responses. The differential chemical composition among MD-A, MD-B, and MD-C, particularly the high arsenic content in MD-A, supports prior evidence that variations in metal and metalloid constituents profoundly influence biological outcomes [39]. The biological impact of MD is determined by both particle load and its chemical and physical properties. This heterogeneity affects particle behavior in the respiratory tract and modulates biological responses like cytotoxicity, cell proliferation, and barrier integrity. An interesting observation from our viability data is that at later time points (48 and 72 h), the cell viability of MD-B and MD-C was significantly higher than that of the controls. This phenomenon differs from the potent mitotic effects of MD-A and can be interpreted as a restorative proliferative response. The initial mild cytotoxicity induced by these particles at 24 h may trigger a compensatory proliferative cascade in viable cells to repair the epithelial monolayer. Alternatively, this increase in CCK-8 signaling may reflect an upregulation of cellular metabolic activity in response to granular stress, rather than a true increase in cell number. Distinguishing these possibilities requires further research, but it highlights the complex, time-dependent adaptive responses of epithelial cells to mineral dust exposure.
This study demonstrated that both the 16HBE14o- and Calu-3 cell lines exhibit adherens junctions and functional TJs, which are essential for maintaining epithelial integrity and controlling paracellular permeability. These intercellular junctions play a crucial role in defending the airway epithelium against environmental antigens that escape mucociliary clearance [50]. In this study, the authors describe the intercellular connections in the 16HBE14o- and Calu-3 epithelial cell lines, emphasizing their significance as robust models for investigating particulate-induced barrier dysfunction. In the present study, the Calu-3 cells were able to form confluent monolayers with high TEER. The progressive increase in TEER, followed by a gradual decline after reaching a peak, aligns with prior studies [25]. Calu-3 cells, which form tightly polarized monolayers with robust TJs, maintained higher TEER after exposure to MD-A, indicating preservation of barrier integrity. We observed notable changes in the fine structure of TJ rings (ZO-1) in Calu-3 cells when compared to other particles (MD-B, MD-C). These changes were marked by more winding cell boundaries and more pronounced breaks forming within the monolayer structure. The TJ abnormalities in Calu-3 cells may stem from the less regular membrane surface topology of these cells as compared to 16HBE14o- cells. Alternatively, the irregularities may indicate the formation of aberrant TJ strands. In addition to MD-A, silica exposure resulted in significant reductions in TEER, indicative of compromised cellular barrier function, reduced the expression of ZO-1 while leaving the cell boundary structures unchanged. In contrast, the 16HBE14o- cells exhibit significantly lower TEER values when cultured under ALI conditions. This may result from the increased total perimeter length of individual cells within the monolayer, which inversely correlates with TEER due to its dependence on junctional organization. 16HBE cells, the TJs display less complex interdigitated patterns and exhibit more diffuse junctional margins compared to those in Calu-3 cells. Furthermore, the presence of arsenic in tin MD adds another dimension of complexity to our understanding, as arsenic exposure is known to trigger a series of intracellular events.
E-cadherin, a central component of adherens junctions, is essential for cell–cell adhesion and for preserving epithelial identity [51]. In this article, the expression of E-cadherin in both cell types formed nearly continuous rings and was primarily localized at the edges of each cell. In 16HBE cells, E-cadherin showed a slight degree of diffuse cytoplasmic distribution. Exposure to silica particles led to a substantial decrease in E-cadherin expression in Calu-3 and 16HBE cells, whereas other particles did not cause any significant changes. In line with the present results, Chuang et al. reported that PM 2.5 from Chinese urban haze events suppressed E-cadherin expression in murine lungs and provoked inflammatory responses [52]. Their study suggested that compromised intercellular adhesion could enhance recruitment of Th2 cells, potentially contributing to airway inflammation. Airway mucus serves as both a physical and biochemical interface between the respiratory epithelium and the external environment, propelled by ciliary motion to remove inhaled particulates [53]. In addition to forming a mechanical shield, epithelial cells secrete mucus containing various protective molecules that trap airborne particles [54]. In our study, MUC5AC expression was detected in both cell types, and exposure to four distinct particulate matters significantly increased MUC5AC protein secretion, which was correlated with elevated MUC5AC mRNA expression levels. Research has shown that fine particulate matter (with a diameter below 2.5 μm) was found to induce MUC5AC mRNA expression in the trachea of mice after 48 h of exposure and both MUC5AC mRNA and protein expression in human bronchial epithelial cells (HBEC) after 24 h of exposure [55]. Moreover, we found that MD particles, which fall within the fine particulate matter range, can increase MUC5AC expression in Calu-3 and 16HBE cells cultured at the ALI. Our findings related to MUC5AC mRNA upregulation further point to potential alterations in mucin production, which could have implications for airway clearance mechanisms following long-term exposure.
Beyond its barrier role, the airway epithelium also orchestrates innate immune responses. Epithelial cells express pattern recognition receptors (PRRs) that detect inhaled pathogens and irritants, initiating cascades that release proinflammatory cytokines, chemokines, and alarmins. These signals recruit and modulate immune cells such as dendritic cells, T cells, and B cells, and activate acute inflammatory pathways [56]. Inhalation of ambient particulates has been shown to enhance epithelial production of cytokines, including IL-1, IL-6, IL-8, IL-25, IL-33, GM-CSF, and TNF-α [57, 58]. The innate immune system thus represents the first defense against airborne insults, with inflammation being the primary short-term toxicological outcome of particle exposure [59]. In our study, all four particle types, when administered via suspension to an in vitro airway model, triggered varying degrees of cytotoxicity and proinflammatory responses. The differences in results highlight the distinct influences of the intrinsic physicochemical properties of particles on biological responses. The significant upregulation of early proinflammatory markers, particularly IL-1β, in cells exposed to MD-A after 24 h indicates that even MD particles that enhance cell viability may simultaneously trigger inflammatory pathways. This duality has been noted in previous research on environmental particulates, where exposures can lead to both proliferative and proinflammatory signals [60]. A key finding of this study is that MD-A induced significant inflammatory responses mainly at the highest exposure level, while low and medium doses were associated with heterogeneous or inconsistent changes. This indicates a threshold-like effect in both 16HBE and Calu-3 cells, whereby only high levels of arsenic-containing dust reliably activate cytokine and mucin gene expression. Importantly, Calu-3 cells also demonstrated clear upregulation of TNF-α and MUC5AC at 10 µg/cm², confirming that the absence of responses at lower doses does not imply reduced sensitivity but rather reflects non-linear dynamics in dose–response relationships. Such a pattern may arise from cellular stress adaptation at sub-threshold exposures, which becomes overwhelmed at higher particle burdens, resulting in a coordinated proinflammatory output. Compared with MD-B and MD-C, which caused less consistent transcriptional alterations, MD-A therefore represents a unique hazard due to its high arsenic content and its dual ability to enhance proliferation and drive inflammatory signaling at high concentrations. From an occupational health perspective, these results highlight the disproportionate risk associated with peak exposures to arsenic-rich dusts and underscore the importance of controlling not only chronic but also acute, high-level exposure scenarios in mining environments.
Rapid cell renewal involves dynamic remodeling of intercellular junctions [61]. As cells divide and integrate into monolayers, existing tight and adherent junctions must be temporarily disassembled and reassembled. This process may manifest as the TEER fluctuations we observed, with more tortuous, seemingly discontinuous ZO-1 staining, which reflects a highly dynamic rather than statically damaged epithelial layer. However, it is also crucial to consider the direct effects of arsenic on connexins. Arsenic and its compounds are known to induce oxidative stress and disrupt cell signaling pathways that directly regulate the integrity and localization of TJ proteins such as ZO-1 and occludin [62, 63]. Therefore, the observed changes are likely the result of a combination of increased cell proliferation and arsenic-mediated direct toxicity to the junction complex. Interestingly, our data show that MD-A exposure (especially after 24 hours) resulted in higher TEER values than other particulate matter. This finding is not entirely consistent with the simple model of proliferation-induced instability, which is often associated with a decrease in TEER [64]. This suggests a more complex reaction in which arsenic may paradoxically trigger an initial ‘overbarrier’ or compensatory tightening effect while the cell layer is in a proliferative state. Additional studies will be necessary to examine those effects.
The purpose of this study was not to compare the differences and advantages between immersion exposure and ALI exposure. Moreover, the exposure concentration of particulate matter in the medium of the immersion experiment is difficult to convert into the mass µg/cm2 deposited on the cell surface of ALI exposure. Specifically, immersion exposure can introduce significant uncertainty in dosimetry. Unlike the precise, real-time quantification of cell deposit mass achieved by QCM in our ALI setup, particles in liquid media undergo complex and unpredictable agglomeration and sedimentation. It is particularly problematic for dense, irregularly shaped mineral dusts, making the actual cell dose unknown and affecting the accuracy of dose-response assessments. In addition, cell physiology is very different. Cells grown in water, especially Calu-3, are unable to form a fully differentiated, polarized monolayer with strong barrier function and mucus-producing capacity. Therefore, the core key endpoints of our study, such as barrier integrity (TEER and ZO-1) and mucin expression (MUC5AC), are insignificant in underwater systems and are fundamentally altered. Finally, the particle-cell interface is non-physiological in immersion cultures, as the particles inevitably acquire a “protein corona” from serum-containing media, which can mask or alter their intrinsic surface reactivity and biological effects.
A key aspect that translates our in vitro findings is the correlation of applied concentrations with real-world occupational exposure scenarios, estimated based on established occupational exposure limits (OEL). Assuming a typical OEL of respirable mineral dust (e.g., 5 mg/m³) and a worker’s daily inhalation of approximately 10 m³, the total inhaled mass would be approximately 50 mg/day. Considering that the tracheobronchial region has a deposition fraction of 10%-30% and its surface area is approximately 2000 cm², the estimated daily deposition dose will be in the range of 2.5–7.5 µg/cm². Our experimental doses range from 1 to 10 µg/cm², which is in good agreement with the estimated daily occupational exposure, suggesting that our findings are highly relevant for assessing acute health risks to miners. This real-world dosimetry enhances the potential of our data to inform targeted interventions in occupational settings.
Despite these advances in our research, several limitations warrant discussion. First, the study was conducted using two immortalized human airway epithelial cell lines. Although 16HBE and Calu-3 provide valuable insights into respiratory epithelial responses, they do not fully replicate the multicellular complexity of the human lung. In vivo, interactions among epithelial cells, macrophages, fibroblasts, and endothelial cells play crucial roles in mediating responses to particulate exposures. Future studies should incorporate co-culture or three-dimensional lung models to better mimic the in vivo environment. Second, the biological endpoints measured in this study were largely related to cellular viability, proliferation, barrier function, and inflammatory signaling. While these markers are essential for understanding acute responses, they do not capture the full spectrum of potential adverse outcomes, such as oxidative stress, genotoxicity, or fibrotic responses. Future studies could explore additional parameters to gain a finer understanding of the toxicity of MD. Additionally, while our experiments were performed under controlled single exposures for durations up to 72 h, the chronic effects of repeated or prolonged exposure remain to be determined. Another limitation of this study is that we only assessed the inflammatory response at the mRNA level. Although gene expression is an early and sensitive indicator of cellular response, future studies should be validated at the protein level by technologies such as ELISA to confirm whether changes in transcription of these genes can be translated into functional protein secretion. No dose-response relationship was observed for most inflammatory marker expression, with sometimes disproportionate responses to small doses of dust. Although it could suggest the high toxicity of the dust even at small dosages, the confusing results could be from technical issues related to the cell culture and RNA extraction. Additional studies are necessary to determine dose-response relationships. The mineral dust particles studied here were micron-scale rather than nanoparticles, so their properties and focus are different from nanomaterials, which are much larger than the effective detection range of DDLS (usually nanometers to a few microns), and DLS is not an appropriate technique to characterize their size. Therefore, the results may be inaccurate or not provide valuable information. Finally, given the observed proliferative effects of arsenic-laden MD, it will be important to investigate the long-term implications of such responses. While enhanced cell proliferation in the short term may suggest a compensatory survival mechanism, sustained proliferative signaling can be a precursor to neoplastic transformation. Future studies should focus on investigating the underlying molecular mechanisms of arsenic-laden MD exposure, thereby further refining exposure thresholds and protective measures.
In summary, our study demonstrates that the integration of advanced aerosol deposition systems with ALI exposure models provides a robust framework for assessing the inhalation hazards of MD particles. While the ALICE system enhances the relevance of in vitro findings to real-world exposures, the emphasis of our study remains on elucidating the toxicological distinctions between different MD samples and understanding how these differences manifest in epithelial responses relevant to respiratory health. Looking ahead, future work will benefit from expanding these methodologies to include a wider array of biological endpoints and more physiologically complex models. In doing so, researchers will be better positioned to predict the long-term health effects of occupational dust exposures and ultimately inform both regulatory standards and strategies for exposure reduction in mining environments. Future research could also use an interesting strategy of using specific metal chelating agents (such as arsenic). By observing whether the pro-proliferative effect of MD-A is weakened or disappears after chelating metals, it can be more directly demonstrated that arsenic is a key factor in this effect. It could also be used to study complex metal mixtures by removing metals one at a time. In addition to arsenic, other metal components may also contribute significantly to the observed toxic effects. For example, high copper levels in MD-A may exacerbate oxidative stress through the release of copper ions [65]. In MD-B and MD-C, despite the low arsenic content, their higher aluminum content may be responsible for their stronger acute cytotoxicity, as aluminum has been reported to be associated with cell damage [66, 67]. Therefore, the overall toxicity of MD is the result of the complex interaction of all its chemical components, rather than being determined by a single element.

Conclusions
This study employed the ALICE system to enable precise, dose-controlled delivery of tin MD particles, facilitating systematic evaluation of their effects on epithelial cell function. Comparative analysis under identical exposure conditions revealed distinct particle-dependent responses, notably enhanced cell viability and modified inflammatory cytokine secretion triggered by arsenic-bearing dust. These findings underscore the critical need for composition-specific risk assessment strategies to address the unique hazards posed by different dust types. By establishing key dose-response relationships for complex MD particles, this work provides essential data to guide targeted interventions aimed at reducing inhalation risks in occupational settings.

Supplementary Information
Below is the link to the electronic supplementary material.