Cytotoxicity and Genotoxicity of Size-Fractionated Iron Oxide (Magnetite) in A549 Human Lung Epithelial Cells: Role of ROS, JNK, and NF-κB

Abstract

Airborne particulate matter (PM) of varying size and composition is known to cause health problems in humans. The iron oxide Fe(3)O(4) (magnetite) may be a major anthropogenic component in ambient PM and is derived mainly from industrial sources. In the present study, we have investigated the effects of four different size fractions of magnetite on signaling pathways, free radical generation, cytotoxicity, and genotoxicity in human alveolar epithelial-like type-II cells (A549). The magnetite particles used in the exposure experiments were characterized by mineralogical and chemical techniques. Four size fractions were investigated: bulk magnetite (0.2-10 μm), respirable fraction (2-3 μm), alveolar fraction (0.5-1.0 μm), and nanoparticles (20-60 nm). After 24 h of exposure, the A549 cells were investigated by transmission electron microscopy (TEM) to study particle uptake. TEM images showed an incorporation of magnetite particles in A549 cells by endocytosis. Particles were found as agglomerates in cytoplasm-bound vesicles, and few particles were detected in the cytoplasm but none in the nucleus. Increased production of reactive oxygen species (ROS), as determined by the 2',7'-dichlorfluorescein-diacetate assay (DCFH-DA), as well as genotoxic effects, as measured by the cytokinesis block-micronucleus test and the Comet assay, were observed for all of the studied fractions after 24 h of exposure. Moreover, activation of c-Jun N-terminal kinases (JNK) without increased nuclear factor kappa-B (NF-κB)-binding activity but delayed IκB-degradation was observed. Interestingly, pretreatment of cells with magnetite and subsequent stimulation with the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) led to a reduction of NF-κB DNA binding compared to that in stimulation with TNFα alone. Altogether, these experiments suggest that ROS formation may play an important role in the genotoxicity of magnetite in A549 cells but that activation of JNK seems to be ROS-independent.

Nanotoxicity of Inert Materials: The Case of Gold, Silver and Iron

“…In some studies, the mechanism seemed to be responsible for the genotoxicity was the ROS generation (158). In other studies, contradictory results were observed including peroxidase like activity of nanoparticles to reduce oxidative stress (159,160).…”

Evaluation of the toxicity of graphene derivatives on cells of the lung luminal surface

“…Several studies proposed oxidative stress as a key mechanism involved in the toxicity of various nanomaterials [31,[45][46][47] that results from the imbalance between excessive [12,31,40,48], which is based on the conversion of the non-fluorescent 2 0 ,7 0 -dichlorodihydrofluorescein diacetate to the highly fluorescent 2 0 ,7 0 -dichlorofluorescein; the substrate is oxidized preferentially by peroxides [39,49]. GO enhanced the formation of intracellular ROS in both A549 and RAW 264.7 cells at the highest concentration of 12.5 lg/cm 2 up to 3.5-times compared to control cells, already after 30 min (Fig.…”

Genotoxicity and reactive oxygen species production induced by magnetite nanoparticles in mammalian cells

“…Several studies have examined the genotoxicity of nanosized magnetite particles; however, the data about this subject are incomplete and do not give a coherent picture. Konczol et al (2011) reported that treatment with magnetite nanoparticles induced ROS production, DNA damage as measured with the comet assay, and micronuclei (MN) formation in human alveolar epithelial-like type-II cells (A549 cells). On the other hand, they did not cause a significant increase in DNA damage or MN formation in Syrian hamster embryo cells (Guichard et al, 2012).…”

“…Their chemical purities were similar as well (≥ 98% and > 99.5% in experiments by Konczol et al and Guichard et al, respectively). Furthermore, both groups reported that the nanoparticles were mainly present as aggregates or agglomerates in cell culture media, and they were incorporated into the cells (Konczol et al, 2011;Guichard et al, 2012). Thus, further toxicity studies are required to increase our understanding of the toxic potential of magnetite nanoparticles.…”