Particle Preparation
The crystalline silica particles, with a size range of 0.2–0.8 mm, were acquired from Merck company. Subsequently, these particles underwent milling using a ball mill, which operated at 380 V and 0.75 kW. Following the milling process, the particles were sieved to achieve microparticles range of 1–10 µm, as well as nanoparticles with dimensions below 40 nm.
For purification, the particles underwent three rounds of washing with hydrochloric acid (1 M). Each tube was subjected to stirring for a duration of 15 min and subsequently left undisturbed for a sedimentation period of 10 min. Following this, all the tubes were subjected to centrifugation at a speed of 5000 rpm for an interval of 20 min. Subsequently, the particles were subjected to treatment with concentrated nitric acid at a temperature of 60°C for a duration of 20 min, three times. Finally, the particles were neutralized using deionized water and dried at a temperature of 40°C for a period of 2 h. The particles were subjected to ICP-OES analysis to determine any metal impurities present, while XRD was employed to assess the silica component within the particle contents.
Cell Culture and Exposure
The cell line A549, derived from human lung epithelial cells, was procured from the Iranian Biological Resource Center (IBRC). These cells were cultivated in a T-25 culture flask using the Dulbecco's Modified Eagle Medium (DMEM) culture medium (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Pen-Strep (Sigma, USA). To provide optimal conditions for cell growth, the culture was maintained at a temperature of 37 ºC, with a 5% CO2 concentration in a humidified atmosphere.
Before the commencement of the experiment, cells were seeded into the 98-well plate, allowing for a period of 24 hours for cell attachment. The silica concentrations for C-SiO2 MPs and C-SiO2 NPs (10, 50, 100, and 250 µg/mL) were then prepared in the cell culture medium without FBS and suspended through the process of ultra-sonication, after which they were gently added to each well. It is important to highlight that the experiments were conducted in triplicate, with appropriate control groups being included. The duration of particle exposure for all experiments was either 24 or 72 h.
Cell Viability Estimation
To conduct the thiazolyl blue tetrazolium bromide assay (MTT), a total of 10,000 cells were introduced into the 96-well plate a day before the commencement of the experiment. The cells were subsequently subjected to varying concentrations (0, 10, 50, 100, and 250 mg/mL) of both C-SiO2 MPs and C-SiO2 NPs. Following a treatment duration of either 24 or 72 h, the wells were thoroughly rinsed with phosphate-buffered saline (PBS) on two separate occasions. Next, 100 mL of MTT solution, prepared at a concentration of 5 mg/mL using Sigma (USA) as the source, was added to each well and allowed to incubate for a duration of 4 h at 37 ºC. Once the MTT solution was removed to facilitate the dissolution of formazan crystals, 100 mL of Dimethyl sulfoxide (Merck, Germany) was gently introduced into each well and mixed in the absence of light for a period of 20 min at room temperature. Subsequently, the wells were analyzed using a plate reader (BioTek Instruments, USA) at a wavelength of 580 nm.
BMD estimation
The BMD evaluation employed the EPA benchmark dose software, version 3.2. The viability of the cells served as the response parameter. However, a continuous response method was implemented to estimation the BMD and BMDL. Various mathematical models, including Exponential 2, Exponential 4, Hill, Polynomial Degree 4, Polynomial Degree 3, Polynomial Degree 2, Power, and Linear, were utilized for the dose-response evaluation. The BMD level was calculated by selecting the response with one standard deviation from the control. The appropriate model was chosen based on the criteria of goodness of p-value, χ2-scaled residual values, Akaike Information Criterion (AIC), and ocular inspection.
BMD extrapolation from A549 to human subjects
The application of the Multiple Path Particle Deposition (MPPD) mathematical model, initially introduced by the Hammer Institute of Health Science in 1995, was justified to transfer data from in vitro experiments to human subjects. Within this mathematical framework, an estimation of the sedimentation and clearing rate of particles in the lungs is made. Consequently, the dose of particles can be determined utilizing Eq. 1.
\({DA}_{in vitro}=\frac{{CV}_{admin}}{{A}_{well}}\) (Eq. 1)
Where DAin vitro represents the mass dose per unit area measured in µg/cm2, C denotes the concentration of the treated solution in terms of BMDL levels measured in µg/mL, V stands for the volume of the treated solution (0.1 ml), and Awell represents the surface area of the in vitro well equal to 0.32 cm2. Finally, the concentration of aerosols deposited in the lung was predicted in terms of µg/m3 using the method described in Eq. 2.
\({D}_{eq}=\frac{{A}_{p}\times {MA}_{in vitro}\times {10}^{6}}{MV\times T\times DE}\) (Eq. 2)
The concentration of Deq is the amount deposited in the human lung (µg/m3). AP, the area of the human pulmonary, is equal to 1020000 cm2. Minute ventilation (MV) is 2000 mL/min (13). The exposure duration, denoted by T (min), is the length of time of the exposure (24 or 72 h). The deposition efficiency, denoted by DE, was 0.1 and 0.23 for C-SiO2 MPs and C-SiO2 NPs, determined using the MPPD model (30).
The concentration of particles deposited in the lungs was converted to airborne concentration with the utilization of appropriate uncertainty factors (Eq. 3). The PED human, which represents the Predicted Exposed Dose in humans, is measured in µg/m3.
The aforementioned factors F1, F2, and F3 play a significant role in this regard.
F1 (Intra-species variation): based on the assumption that the variability in the general population, including children, the elderly, and diseased individuals, is higher than in workers, A default value of 5 for workers was applied (31, 32).
F2 (LOAEL to NOAEL extrapolation): the BMDL10 is considered a LOAEL since the biological effect was a 10% increase in cancer incidence. A maximum factor of 10 is used when deriving an OEL from a LOAEL, instead of a NOAEL (31).
F3 represents the short-term exposure; This factor is assigned a value of 10 (33).
\(PED=\frac{{D}_{eq}}{{F}_{1}\times {F}_{2}\times {F}_{3}}\) (Eq. 3)