Background

Coarse particles are mostly deposited in the upper respiratory tract, whereas fine particles can be inhaled deep into the lung [1]. UFPs could directly injure the lung, inducing lung inflammation or translocation of inhaled particles from lung airspaces into the systemic circulation, eventually reaching other organs [2-5]. A number of studies have investigated the toxicity of ambient particles [6,7]. More recently, nanotoxicology has emerged as a new field for investigating the adverse biological outcomes of nanomaterials [2,8-10]. Because engineered nanoparticles (NPs) are now being produced in huge quantities, increased human and environmental exposure from various mechanisms, such as fugitive emission, accidental spills, and normal usage, is inevitable. Moreover, current environmental laws and occupational health guidelines are based on the nominal chemical composition of the material and seldom specify special standards for ultrafine or nanosized particles. Therefore, the potential occupational health and environmental effects of these nanosized particles are a public health concern [11].

Toxicologists have begun to focus on investigating the toxicological effects of exposure to NPs [10-14]. Because of the larger total surface area to volume ratio, small size, and other physicochemical properties (such as ability to absorb toxic metals and polycyclic aromatic hydrocarbons) of NPs, these particles can display toxicity profiles that are very different from those of larger materials of the same composition [10,15], indeed, NPs have been shown to be more toxic than coarse and fine particles [4,5]. A number of studies have focused on the physicochemical characterization and toxicity of ambient particles [16], and a mechanism through which ambient particle induce toxicity (i.e., the oxidative stress theory) has been established [3-5]. Importantly, some of the procedures and assays that are generally used to assess the adverse biological effects of ambient PM could be applied for the study of engineered NPs [2]. For example, the ability to generate ROS and oxidative injury may provide a paradigm to compare the toxic potential of NPs [14]. However, studies comparing the toxicity of exposure to ambient UFPs and engineered NPs are limited.

In this study, we compared the toxicity of metal oxide NPs (ZnO, NiO, and CeO2), which have been primarily used for industrial purposes, to that of ambient ultrafine, fine, and coarse particles sampled from the atmosphere in Shanghai. These results could provide fundamental data for the development of health risk assessments with respect to exposure to engineered NPs and airborne UFPs.

Results

Physicochemical characterization of size-resolved ambient particles

Mass concentrations of the size-resolved ambient particles

Mass levels of the airborne particles differed according to particle size (Figure 1). The average mass concentrations of coarse particles, fine particles, and UFPs were 95.34 ± 24.92, 77.41 ± 15.6, and 25.03 ± 4.61 μg/m3, respectively.

Figure 1
figure 1

Mass concentration of ambient size-resolved particles collected at SDK site in winter in 2009. The sampling campaign was carried out from November 23 to December 5, 2009. The value of mass concentration of the size-resolved particles was expressed as means ± standard deviations (SDs; n = 5). The error bars indicate standard deviation.

Full size image

Chemical elements in the ambient particles

Next, the mass concentrations of 20 elements, i.e., Si, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, and Pb, in Shanghai size-segregated particles were investigated by PIXE (See Additional file 1: Table S1). The chemical elemental analysis results showed that calcium (2380.9 ng/m3) was the most abundant crustal element in the coarse particles, while Zn (490.48 ng/m3) was the most abundant trace elements in the fine particles. Si (83.66 ng/m3) was the most abundant element among the measured elements in the UFPs.

Microscopic characterization of ambient particles

SEM revealed that particles classified into the different size categories had visibly different attributes. Based on SEM morphological characteristics (Figure 2) and EDX spectra (data not shown), individual particle types in the sampling site atmosphere could be identified as fly ash (Si, Al, and O), soot particles (carbon), regular mineral particles (S, O, Cl, and Na), and unidentified particles.

Figure 2
figure 2

SEM micrographs of size-segregated particles collected in Shanghai suburban atmosphere. (A) coarse particles; (B) fine particles; (C) ultrafine particles; (D) fly ashes; (E) soot aggregates; (F) mineral particles containing N, O, (G) salts (NaCl); (H) and (I), mineral particles containing S. Scar bar: 1 μm.

Full size image

Microcharacterization of the nano-ZnO, -NiO, and -CeO2 particles was performed using TEM. High-resolution TEM images revealed that nano-ZnO had a crystal structure, while nano-NiO and -CeO2 were in an amorphous state (Figure 3).

Figure 3
figure 3

TEM morphological characterization of metal oxide nanoparticles. nano-ZnO had a crystal structure, while nano-NiO and -CeO2 were in an amorphous state.

Full size image

MTT results

In this experiment, due to the very low mass level of UFPs, the mass doses of the ambient UFPs were only 25 and 50 μg/mL. With these concentrations of UFPs, MTT assays were used to evaluate the proliferation of A549 cells treated with ambient size-resolved particles and metal oxide NPs. The MTT assay results showed that cell viability decreased in response to exposure to size-resolved ambient particles and NPs at 25, 50,100, and 200 μg/mL (Figure 4). For example, exposure to the insoluble and soluble fractions of coarse particles at 25 μg/mL yielded cell viabilities of 0.97 ± 0.02 and 0.91 ± 0.01, respectively, cell viability was decreased to 0.7 ± 0.09 and 0.73 ± 0.11, respectively, when the concentration of particles was increased to 200 μg/mL. Correspondingly, exposure to the insoluble and soluble fractions of fine particles decreased cell viabilities to 0.85 ± 0.01 and 0.92 ± 0.06 at 25 μg/mL and 0.6 ± 0.03 and 0.7 ± 0.06 at 200 μg/mL, respectively. Treatment with insoluble and soluble fractions of ambient UFPs at 25 and 50 μg/mL also inhibited the proliferation of A549 cells (0.94 ± 0.01 and 0.86 ± 0.04, respectively). Cell viability induced by NPs decreased in a concentration-dependent manner.

Figure 4
figure 4

Cell variability caused by ambient size-resolved particles and metal oxide nanoparticles ($, p < 0.05, PM 5.6 insoluble(200 μg/ml) vs PM 5.6 insoluble(25 μg/ml); $$$ p < 0.001, PM 5.6 insoluble(100 μg/ml) vs PM 5.6 insoluble(25 μg/ml); # p < 0.05, PM 1.8 insoluble (100 μg/ml) vs PM 1.8 insoluble (25 μg/ml); @ p < 0.05, PM 1.8 insoluble (200 μg/ml) vs PM 1.8 insoluble (25 μg/ml) p < 0.01; *, p < 0.05, PM 5.6 soluble (100 μg/ml) vs PM 5.6 soluble (25 μg/ml);, 〒p < 0.05, Nano NiO (100 μg/ml) vs Nano NiO, △Nano NiO (200 μg/ml) vs Nano NiO (100 μg/ml); △△p < 0.01 Nano NiO(200 μg/ml) vs Nano NiO(50 μg/ml) △△△Nano NiO(200 μg/ml) vs Nano NiO(25 μg/ml).

Full size image

LDH activity of the measured particles

The LDH assay results showed that the different groups of particles induced varying levels of cytotoxicity. Compared with the control group, the LDH activity in cells treated with PM1.8 or NPs was significantly elevated (Figure 5, p < 0.05). Moreover, PM1.8 and NPs exhibited concentration-dependent effects on LDH activity. It was noteworthy that LDH activity induced by PM1.8(soluble fraction) was stronger than that of PM1.8 (insoluble fraction).

Figure 5
figure 5

LDH activity of A 549 cells treated with the panel of measured particles. # p < 0.05 PM1.8(insoluble) vs control; @ p < 0.05 PM1.8(soluble) vs control; $ p < 0.05 PM5.6(soluble) vs control,;& p < 0.05 Nano CeO2 vs control; *p < 0.05 Nano NiO vs control; ▲p < 0.05 Nano ZnO vs control. Values are mean ± SD from three independent experiments.

Full size image

Intracellular ROS

Similar to the results of cell viability and LDH activity, different particles exerted varying effects on intracellular ROS generation (Figure 6). Importantly, all of the particles tested in this study stimulated the induction of intracellular ROS. The highest fluorescent intensity (0.098 ± 0.013) was induced by PM1.8 (soluble fraction with 200 μg/mL), while the lowest fluorescent intensity (0.024 ± 0.003) was induced by ambient UFPs (insoluble fraction with 50 μg/mL).

Figure 6
figure 6

Intracellular ROS in A549 cells exposed to different components of particles, # p < 0.05 as compared to Control; **p < 0.05 as compared to PM 1.8 (soluble). Values are mean ± SD from three independent experiments.

Full size image

Induction of apoptosis in A549 cells exposure to the different particles

Next, we analyzed the induction of apoptosis in A549 cells after a 12-h incubation with the different particle solutions. As shown in Figure 7, all the particle solutions could induce apoptosis in A549 cells. Interestingly, the soluble fractions of ambient particles induced significantly higher percentages of apoptotic cells compared the insoluble fractions and NP solutions.

Figure 7
figure 7

Apoptosis of A549 cells after exposure to 200 μg/ml measured particles for 15 mins. A-ultrafine particles (insoluble fraction); B-fine particles(insoluble fraction); C-coarse particles(insoluble fraction); D-ultrafine particles (soluble fraction); E-fine particles (soluble fraction); F-Coarse particles (soluble fraction); G-Nano CeO2; H-NiO; I-ZnO; J-control. The data was expressed as the mean ± SD of three independent experiments. *p < 0.05 PM1.8(soluble) vs control; **p < 0.01 PM5.6(soluble) vs control; ***p < 0.001 PM0.1(soluble) vs control; $$$ p < 0.001 PM0.1 (insoluble) vs PM5.6(soluble); ### p < 0.001 PM1.8 (soluble) vs Nano CeO; @@ p < 0.01 PM5.6(soluble)vs NiO; △△p < 0.01 PM5.6(soluble)vs ZnO.

Full size image

Distributions of metals in A549 cells

Compared with the control, Fe, Ni, Cu, and Zn exhibited differential distributions in A549 cells exposed to size-resolved ambient particles (Figure 8). The fluorescence intensity of copper (Figure 8-h) in A549 cells was the highest, followed by Zn, Ni, and Fe. For the different sizes of ambient particles (coarse, fine, ultrafine), the four heavy metals exhibited the same trends, with Cu being most abundant (followed by Zn, Fe, and Ni; Figure 8-g, h, j–r). Interestingly, among NPs, the fluorescent intensity of nano-ZnO particles was higher than that of NiO NPs in A549 cells.

Figure 8
figure 8

Distribution of selected metals in subcelluar of A549. A-cells seeded in Si3N4 plate under light microscopy (X 400); B-control (without any particles in cells); C-Nano NiO particles; D-Nano ZnO particles; F to J, distribution of Fe, Ni, Cu, Zn in PM 5.6 respectively; K to N, distribution of Fe, Ni, Cu, Zn in in PM1.8 respectively; O-R, distribution of Fe, Ni, Cu, Zn in in PM0.1 respectively. Scar bar: 10 μm. Colour bar indicated different fluorescent intensity induced by metals in the A549 cell.

Full size image

Discussion

Accumulating epidemiological and toxicological evidence has shown that there are important health risks associated with exposure to engineered NPs and ambient UFPs [9,15]. Indeed, some UFPs can be found in the heart, lungs, blood vessels, and other organs after exposure to ambient air containing nanomaterials [28], with modifications. A drop of A549 cell solution containing 10% FCS was removed onto a sterilized Si3N4 crystal plate (attached to the bottom of a 24-well plate) using of pipette, and 5 mL DMEM was added into the wells. The plate was then incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. After the Si3N4 plate (with cells attached) was washed with D-Hank’s three times, 5 μL of the particle solution (ambient particles, NPs; 25 μg/mL) was added into the wells. Then, the Si3N4 plate was kept in the incubator for 4 h. The plate was washed with D-Hank’s, and the cells were fixed on the Si3N4 with alcohol (95%).

The distribution of metal in the cells was analyzed with SR-μXRF on a beamline BL15U instrument at Shanghai Synchrotron Radiation Facility (Shanghai, China) (Qiu et al.) [29]. The energy of the storage ring was 3.5 GeV and the beam current was 200–300 mA. Continuous synchrotron X-rays were monochromatized by an Si (111) double crystal. A monochromatic X-ray beam with a photon energy of 10 keV was used to excite the samples. The cross-section of the beam irradiating on the samples was adjusted to about 2 × 2 μm2. The sample was placed at a 45° angle to the incident X-ray beam, and X-ray fluorescence was detected with a 50 mm2 silicon drift detector (Vortex, USA) oriented at a 90° angle to the incident beam. A light microscope was coupled to a computer for sample viewing. The sample platform was moved by a motorized x-y map** stage. The distributions of selected metals (Ni, Fe, Cu, and Zn) in the cells were continuously scanned at a step of 2 μm for both the x and y directions. Each spot was irradiated for 100 s. The data were analyzed by Plot software, and the differential metal distribution map was exported.

Statistical analysis

The data for mass concentration of ambient particles was analyzed by Excel and expressed as means ± standard deviations (SDs; n = 5). The MTT assays, LDH assays, and intracellular ROS data were analyzed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA), and data were expressed as means ± SDs. Statistical significance was determined by using one-way analysis of variance (ANOVA). Differences with p values of less than 0.05 were considered significant.

Supporting information

More details on mass level of chemical elements in the size-resolved particles were listed in supplementary table.