Surface chemistry alterations from UV radiation.
Compared to the pristine polystyrene microspheres (Figure 1A), UV irradiation for 5 weeks caused a visible alteration to the surface of the polystyrene microspheres, as observed by scanning electron microscopy (SEM) (Figure 1B); notably the microspheres appeared otherwise intact. The primarily smooth and rounded surfaces of particles transformed under UV light into rough and deformed surfaces with no detectable changes in the size of particles (Figure 1A, B). Raman spectroscopy revealed a consistent polystyrene spectrum with no detectable changes between aged and fresh microspheres. (Figure 1C, D).
Additional analyses were conducted using X-ray photoelectron spectroscopy (XPS) to identify any weathering effect on the organic functional groups by measuring the signal of C 1s on the near-surface region of the fresh and aged microspheres (Figure 1E). Curve fitting of high-resolution C 1s spectra reveals the photooxidation of UV-irradiated polystyrene microspheres at the top near-surface region by showing changes in peak intensity of polar functional groups compared to fresh microspheres. Overall oxygen content, measured by XPS, was increased roughly 10% on both sizes of polystyrene microspheres exposed to UV-light (Figure 1F). Increased single and double carbon-oxygen bonds were also detected in the 5 µm microspheres after UV-aging with an increase in the relative percent of C-O groups of 95% and 32% in 1 and 5 µm microspheres, respectively (Figure 1G). Similarly, carboxyl (COOH) groups increased by 75% and 137% in 1 and 5 µm microspheres, respectively. A different pattern was observed for the non-polar bonds C-C, C-H % by showing a decrease of the corresponding peaks intensity in high-resolution C 1s spectra after UV-aging. It should be noted that there was also a decrease in the relative percent of C=O functional groups after UV-aging for both 1 µm and 5 µm microspheres. Meides et al. reported an increase in ketone groups only after a longer exposure of PS particles to UV radiation (>2000 hours)[24]. The non-detectable changes in functional chemistry by Raman spectroscopy (which has a depth resolution about 1 micron) confirm that the increase in polarity is limited to the top 5-10 nm of the near-surface region of the particles, which could only be detected by XPS analysis. Our microscopic and spectroscopic findings confirm that photoaging of 5 weeks causes physical alteration of the surface of the microspheres and increases the polar groups of polystyrene chains on the near-surface region that may affect the interactions of particles with mammalian cells. Given that the surface chemistry is altered after UV-aging, we set out to understand if this change would impact the lung epithelium.
Polystyrene microspheres affect cellular morphology and metabolism.
To understand how both pristine or photoaged microplastics impacted epithelial homeostasis, we applied high-content analysis to examine effects of fresh or UV-aging, dose (1, 3, 10 and 30 µg/ml), and size (1 and 5 µm microspheres) at multiple time points (24, 48 and 72h) on A549 cells. Specifically, we quantified nuclei number and nuclear area, and assessed nuclear shape and total intensity. We also assessed DNA damage using the DNA double-strand break marker phosphorylated H2A histone family member X (γ-H2AX), and epithelial barrier function using FAK and F-actin in the individual cells. We used the heavy metal cadmium (Cd) at 2.5 mM as a positive control toxicant.
Overall DNA intensity measures were unchanged by microsphere treatment (Figure S1)and there was no significant alteration of γ-H2AX intensity, suggesting no double-strand DNA breaks (Figure S2). Cell numbers per well were significantly altered by microplastics treatment. Pristine polystyrene microspheres, at both 1 µm and 5 µm sizes, caused a dose-dependent increase in cell numbers per well compared to untreated cells (Figure 2). On the other hand, aged microsphere exposure led to increased cell numbers at the lowest dose (1 µg/ml), but cell numbers per well decreased at higher doses. These dose-related patterns, for both pristine and UV-aged microplastics were evident and consistent at 24h, 48h, and 72h. It is possible that the microplastics exhibit a biphasic effect on cell growth that culminates in loss of cells due to cytotoxicity, and that the aged microplastics exhibit a dose-response relationship that has shifted due to more potent toxicity.
Nuclear size was significantly reduced in A549 cells treated with either size of polystyrene microspheres across all doses (Figure 3A). We further quantified FAK at 48h, and showed that treatment of microplastics at both bead size dose-dependently reduced the intensity of FAK (Figure 3B). Changes in nuclear shape can occur through alterations in nuclear lamina or the cytoplasm and cellular morphology [28]. Since F-actin is involved in maintaining cell structure and rigidity, we sought to determine if microplastics caused morphological changes in A549 cells by examining cytoskeleton F-actin via high-content analysis. F-actin was evenly distributed across the whole well in control (untreated) cells; however, treatment with UV-aged microplastics resulted in disruption of f-actin structure (Figure 4). Green microparticles indicated that these particles were enwrapped inside of the f-actin (white arrow), while the blue particles indicated these particles were outside of the cell body. We further examined the effects of microplastic on formation of alpha-tubulin (green) and the expression of FAK (red, Figure 5). Using Cd as a positive control to show cellular disruption, we observed disrupted FAK expression, which increased stress fiber formation. Treatment with 30 µg/ml of UV-aged microplastic also disrupted the FAK and increased the stress fiber formation of alpha-tubulin, similar to what was observed for Cd treatment.
Stress can cause changes in cytoskeletal architecture, resulting in changes in cellular metabolism [29]. Therefore, we utilized an assay to measure the proton efflux, which is derived from both glycolysis and mitochondrial / tricarboxylic acid cycle activity to determine the rate of glycolysis in A549 cells after overnight exposure to pristine or photoaged microplastics at various concentrations (0, 1, 10, or 20 µg/ml). Briefly, the inhibition of mitochondrial function via rotenone and antimycin A enables the calculation of mitochondrial-associated acidification (mitochondrial oxygen consumption rate, mitoOCR). Subtraction of mitoOCR from the total Proton Efflux Rate (PER) enables the calculation of PER derived from glycolysis, denoted as the Glycolytic Proton Efflux Rate (GlycoPER). All doses and status of microplastics increased basal glycolysis in A549 cells compared to unstimulated A549 cells (Figure 6). Taken together, the change in cellular metabolism along with the alterations in the cytoskeleton architecture suggest microplastics are damaging the cell membrane.
Polystyrene microspheres-induced cellular stress causes G2 arrest.
As shown in Figure 7, cell population in S and G2 phases significantly decreased at 48 h and 72 h compared to 24 h, since cells reached 100% confluence with minimum DNA synthesis and cell proliferation. Treatment with cadmium significantly decreased the S populations at 48 and 72 h. We found statistical significance (p<0.001) of a four-way factorial model analysis of cells in S population versus concentration (1-30 mg/ml), time (24, 48, and 72h), status (Fresh and UV-aged) and bead size (1 and 5 mm). Two-way ANOVA comparison at each time point showed that there are significant differences in cell population in S phase between the fresh and UV-aged microplastics at all three timepoints. Significant increase of cells in S phase at the dose of 1, 3 and 10 µg/ml of UV-aged microplastics of bead size of 1 µm at 24h, and decrease at the dose of 30 µg/ml of UV-aged microplastic of bead size of 5 µm. At 48h, regardless of bead size, all doses of fresh or UV-aged microplastic except 30 µg/ml of UV-aged microplastics increased the cells in S phase. At 72 h, regardless of bead size, all doses of fresh or UV-aged microplastic except 30 µg/ml of UV-aged microplastics with a bead size of 5 µm increased the cells in S phase. Increases of cells in G2 phase after treatment with cadmium were observed in 24 and 48h. Treatment of UV-aged microplastic at both 1 and 5 µm increased the numbers of cells in G2 phase at 24h and 48h at all doses (except one dose at 1 µg/ml of 1 µm bead size at 48h). Significant increases of cells in G2 phases at 72h were only observed at UV-aged microplastics at 10 and 30 µg/ml for both 1 and 5 µm bead size. Changes in G2 phase in the fresh microplastics were only observed in 10 and 30 µg/ml in the bead size of 1 µm.
UV-aged polystyrene microspheres impact cell monolayer integrity and wound healing.
Confluent A549 cells were treated with fresh and photoaged microspheres (1 µm, 5 µm or a mix of both sizes) at 1 or 10 µg/ml. Electrical resistance was measured across the A549 monolayer as an index of barrier integrity. After the initial dosing, which included a change of media and a brief increase in resistance, all cells treated with fresh polystyrene microspheres at 1 or 10 µg/ml trended towards a lower resistance level compared to untreated controls (Figure 8). The smaller particles induced a greater loss of resistance. A greater loss of monolayer resistance was observed in cells treated with photoaged microspheres than in cells treated with fresh polystyrene microspheres. This suggests that surface chemistry changes could affect biocompatibility.
Another integral part of barrier integrity is the ability to heal after an insult/injury. Therefore, stably confluent cells were treated with a brief electric current [30] to eliminate cells covering the electrodes on the microwells, following 24h incubation with polystyrene microspheres or vehicle. The electric current led to a substantial drop in resistance in all wells, which recovered over the next 5 hours as cells regrew over the detection electrode (Figure 9). Fresh 1 µm polystyrene microspheres at the low dose (1 µg/ml) only exhibited a modest reduction in wound healing, with 5 µm microspheres having no effect; the higher dose (10 µg/ml) potentiated this effect. UV-aging of the plastics exacerbated the wound recovery deficits at both doses. The high-dose mixture of 1 µm and 5 µm photoaged plastics reduced regrowth by 65%. Taken together, our data suggest the exposure of the lung epithelial cells to pristine or photoaged microplastic could prove detrimental to airway function and injury recovery.