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Cytotoxicity of copper (II) oxide nanoparticles in rat intestinal cells: effect of simulated gastrointestinal fluids and generation of oxidative stress
Citation:
Henson, T., J. Navratilova, K. Bradham, K. Rogers, AND M. Hughes. Cytotoxicity of copper (II) oxide nanoparticles in rat intestinal cells: effect of simulated gastrointestinal fluids and generation of oxidative stress. Society of Toxicology, San Antonio, Texas, March 11 - 15, 2018.
Impact/Purpose:
There is a potential for human oral exposure to copper (II) oxide nanoparticles following accidental or intentional ingestion, hand-to-mouth activity, or mucociliary transport following inhalation. The objective of this study was to assess the cytotoxic effects of copper oxide nanoparticles (size <50 nm) in rat intestinal cells following pretreatment with simulated gastrointestinal fluids. In addition, a mode of action of the cytotoxicity, generation of oxidative stress, was also assessed.
Description:
Metallic oxide nanoparticles (NPs) have a variety of applications in industry, medicine and commercial products. Exposure to NPs can occur by inhalation, dermal contact and oral ingestion. We have previously reported on the dose- and time-dependent cytotoxicity of CuO NPs (size < 50 nm) in rat intestinal cells (IEC-6) from the aspect of oral ingestion. This study assessed the effect of pretreating CuO NPs (1 mg/ml) with simulated gastrointestinal (GI) fluids (pepsin at pH 2 to 6, pancreatin at pH 7, bile salts at pH 7; incubated sequentially) on cytotoxicity in rat intestinal cells (IEC-6). The treated NPs were isolated by ultracentrifugation, suspended in media and probe sonicated before dosing the cells. Cells were incubated for 24 hr with the treated NPs (0.1 – 100 ug/ml) and cytotoxicity was assessed using a colorimetric method that measures mitochondrial activity (MTS assay). The zeta potential (ZP) and hydrodynamic diameter (HD) of similarly treated CuO NPs were measured after each incubation step. The ability of non-treated or pristine CuO NPs to generate oxidative stress in the cells was also assessed. Following a 4-hr exposure to CuO NPs (0.1 – 100 ug/ml), H2O2 and glutathione (GSH) were quantitated in the cells by a bioluminescent assay. Pretreatment of CuO NPs with GI fluids increased the cytotoxicity by 30% at 5 ug/ml relative to non-treated NPs. No differences were observed at the other concentrations. The lowest ZP (-3.4 mV) and highest HD (5104 nm) of the NPs was observed after incubation with pepsin at pH 2. Raising the pH stepwise one pH unit to pH 6 and incubating with pepsin increased ZP and decreased HD. Consistent changes in ZP and HD were not observed at the other two step of pretreatment. The 24-hr exposure of IEC-6 cells with pristine CuO NPs showed a dose-dependent increase in cellular H2O2 and a concomitant decrease in GSH. In summary, treating CuO NPs with simulated gastrointestinal fluids can alter their ZP and HD, but the modifications result in minimal alteration in cytotoxic effects of the particles. Pristine CuO NPs can generate oxidative stress within the cells, which is a potential mechanism of action for these particles. Consideration of the exposure scenario and mechanism of action of NPs are important aspects for the risk assessment of this emerging material. (This abstract does not represent US EPA policy.)