Biological effects of inhaled hydraulic fracturing sand dust. III. Cytotoxicity and pro-inflammatory responses in cultured murine macrophage cells

Abstract

Cultured murine macrophages (RAW 264.7) were used to investigate the effects of fracking sand dust (FSD) for its pro-inflammatory activity, in order to gain insight into the potential toxicity to workers associated with inhalation of FSD during hydraulic fracturing. While the role of respirable crystalline silica in the development of silicosis is well documented, nothing is known about the toxicity of inhaled FSD. The FSD (FSD 8) used in these studies was from an unconventional gas well drilling site. FSD 8was prepared as a 10 mg/ml stock solution in sterile PBS, vortexed for 15 s, and allowed to sit at room temperature for 30 min before applying the suspension to RAW 264.7cells. Compared to PBS controls, cellular viability was significantly decreased after a 24 h exposure to FSD. Intracellular reactive oxygen species (ROS) production and the production of IL-6, TNFα, and endothelin-1 (ET-1) were up-regulated as a result of the exposure, whereas the hydroxyl radical (^.OH) was only detected in an acellular system. Immunofluorescent staining of cells against TNFα revealed that FSD 8 caused cellular blebbing, and engulfment of FSD 8 by macrophages was observed with enhanced dark-field microscopy. The observed changes in cellular viability, cellular morphology, free radical generation and cytokine production all confirm that FSD 8 is cytotoxic to RAW 264.7 cells and warrants future studies into the specific pathways and mechanisms by which these toxicities occur.

Keywords: Cytotoxicity; Fracking sand dust; Inflammation; Macrophages; Occupational exposure.

Fig. 1.

Size and surface area distribution profile of FSD 8 in suspension. Average particle size ± standard error, along with mode, particle concentration, and distribution of particles according to size, are shown. D₅₀ indicates the percentage of particles that are below the 50th percentile, which gives an indication of the distribution of particle sizes within the sample.

Fig. 2.

Effect of FSD 8 on cell viability. At 24 h, a significant decrease in cellular viability was observed in the 5 mg/ml treatment group when compared to PBS controls. The viability of cells treated with 5 mg/ml FSD was decreased significantly at 24 h when compared to 4 h. ^aFSD 8 vs. PBS; ^b4 h vs. 24 h (within dose); ^cmg/ml vs. 5 mg/ml.

Fig. 3.

˙OH production stimulated by FSD 8 in an acellular system. Signal intensity (peak height) was used to measure by ESR the relative amounts of ˙OH produced. A) Fenton-like reactions were carried out in an acellular system using FSD 8 suspensions. B) Representative spectra is shown. ^aFSD 8 vs. PBS.

Fig. 4.

Intracellular ROS production in FSD 8 supernatants. At the 2 h and 4 h time points, both concentrations of FSD 8 produced significant amounts of intracellular ROS when compared to PBS. ^aFSD vs. PBS.

Fig. 5.

FSD 8 causes DNA damage. A) At 0.5 mg/ml and 1.0 mg/ml, FSD 8 produced significantly greater and dose-dependent damage to DNA when compared to PBS controls. B) Micrographs are representative images of RAW 264.7 cells treated with FSD 8 at the 24 h time point. ^aFSD 8 vs. PBS; ^aFSD vs. Cr(VI); ^c0.5 mg/ml vs. 1.0 mg/ml.

Fig. 6.

Time- and dose-dependent production of TNFα and IL-6 in response to FSD 8 treatment. (A) At 24 h, RAW 264.7 cells treated with 5 mg/ml FSD 8 produced significantly greater amounts of TNFα when compared to PBS controls, and also when compared to treatment with 1 mg/ml FSD 8. TNFα was not up-regulated at the 4 h time point by treatment with either dose. ^aFSD 8 vs. PBS; ^b4 h vs. 24 h (within dose); ^c 1 mg/ml vs. 5 mg/ml (within time point). (B) Peak IL-6 production occurred at 4 h. At 4 h, both the 1 mg/ml and 5 mg/ml treatment groups caused significant production of IL-6 when compared to controls. By 24 h, the IL-6 returned to near-baseline levels. ^aFSD 8 vs. PBS; ^bmg/ml vs. 5 mg/ml (within time point); ^c 4 h vs. 24 h (within dose).

Fig. 7.

Increase of ET-1 levels following incubation with FSD 8. Both doses of FSD 8 caused a significant increase in ET-1 production by cells at 24 h when compared to PBS. ^aFSD 8 vs. PBS.

Fig. 8.

Cellular blebbing and stress in response to treatment with FSD 8. All images were taken at 20× magnification unless noted otherwise. Row A, RAW 264.7 cells exposed to PBS for 24 h. Row B, Cells treated with FSD 8 display irregular morphology, granulated nuclei, and cellular blebbing (red arrow). Row A and B, bar = 25 μ. Row C, bar = 10 μ.

Fig. 9.

RAW 264.7 cells phagocytize FSD 8. Anuclear cell fragments and nuclear condensation were observed (red arrow), along with plasma membrane blebbing (white arrow) in cells exposed to FSD 8 for 24 h. Bar = 20 μ.