The impact of nanoparticles on the proteome of cultured human cells

Abstract

Living organisms are constantly being exposed to nanoparticles (NPs) in the environment via air, water, soil. Routes of exposure are usually in the form of industrial, occupational exposure, as well as therapeutic applications. This exposure could result in toxicity with potential harmful effects. The toxicity of nanoparticles depends on various factors such as surface interaction, shape, size, composition, aggregation and interaction with various cellular components. Nanotoxicity refers to the possible harmful effects of environmentally generated and man-made nanoparticles on biological and environmental system. Assessing potential toxicity is vital for the probable use and safety of nanoparticles as well as understanding the routes of entry into organisms and their mechanism of action. Proteomics is a developing field of science that is being explored to understand protein composition, structure and interaction at the cellular level. This helps in detecting the presence, quantity, alteration and regulation of proteins within the biological system. The proteome analysis brings an additional information as it enables measurement of whole protein (enzyme) expression levels, facilitating the construction of metabolic pathways and biomarker discovery for early disease diagnosis. Essentially, proteomic analysis reveals the consequence of stress on metabolic pathways necessary to maintain the energy homeostasis within the cells. Interaction mechanisms between nanoparticles and biological systems in relation to proteomics are not yet fully understood. In addition, scientific knowledge about the mechanisms involved in nanoparticle–cell interaction has been accumulating in recent years and have shown that cells readily take up nanoparticles via either active or passive mechanism. Hence this research work seeks to investigate the effect of nanoparticles on the proteomic profile of human epithelial cells so as to improve the application and uses of nanoparticles in delivery, diagnostic, imaging, therapy and environmental remediation. The nanoparticles selected for this study are silver (Ag) and titanium dioxide (TiO2) nanoparticles with the aim of evaluating the possible harmful effects, inflammatory modulation, oxidative stress and uptake mechanism. The colon cancer epithelial cell line (Caco-2) was selected as a model for intestinal epithelium in this study for evaluation of the possible toxicity of the selected nanoparticles. The nanoparticles parameters evaluated included hydrodynamic size and zeta potentials. The exposure of Caco-2 cells to the respective nanoparticles assessed cell viability, proteome profile analysis, cell stress, inflammatory biomarkers, anti-angiogenic properties and uptake mechanisms. The first set of objectives of this study was to elucidate the physiological effects that various media would have on AgNPs and TiO2NPs hydrodynamic size and zeta potential. Thereafter the effects of these characterized nanoparticles on cell viability, cell stress, inflammatory biomarkers and antiangiogenic properties on Caco-2 cells were evaluated. The results showed that the AgNPs were stable in physiological media, based on hydrodynamic size and zeta potential over the time period assessed 0 hour, 24 hours, 7 and 14 days. Whereas, the TiO2NPs sizes increased over time, while zeta potentials were stable. The cytokine proteome profile membranes assessed for the AgNPs mostly revealed the same proteins, with the exception of activin A and dipeptidyl peptidase IV (DDPIV) which was only present in the control group. The intensity of certain proteins decreased in the presence of the IC50 value in comparison to the control. These proteins include: angiopoietin2, endostatin and platelet-derived growth factor AA (PDGF-AA). Conversely, persephin was slightly upregulated in the presence of 100 g/ml AgNPs in comparison to the control group. The AgNPs are cytotoxic to Caco-2 cells at high concentrations (IC50 = ± 100 g/ml) and induced cell stress biomarkers at concentrations > 6.25 g/ml AgNPs. The AgNPs modulated the inflammatory cytokine IL-8 and reduced IL-6 production in a dose dependent manner. Also, possible antiangiogenic markers of AgNPs were identified: angiopoietin-2 and PDGF-AA. The TiO2NPs proteome profile showed that the control (0 g/ml TiO2NPs) and NOAEL (100 g/ml TiO2NPs) supernatants assayed for potential angiogenic biomarkers revealed the same proteins, except persephin which was only evident in the control membrane. Based on the intensity of the dot, the protein dipeptidyl peptidase IV (DPPIV) and endostatin was more prominent on the NOAEL membrane. However, platelet derived growth factor AA (PDGF-AA) and angiopoietin-2 was more noticeable to cells exposed to control concentration. The level of inflammatory biomarkers was not affected. Subsequently, anti-angiogenic properties were exhibited when exposed to the no-observed-adverse-effect level (NOAEL) concentrations of the TiO2NP. The TiO2NPs induced cell stress biomarkers, which could be attributed to the NPs not being cytotoxic. The last objective of this research was to evaluate the uptake mechanism effects on the toxicity of nanoparticles through the cytotoxic and/or inflammatory pathways. Different pathway inhibitors were used such as Amiloride chloride for (micropinocytosis), ammonium chloride (phagocytosis), chlorpromazine (clathrin mediated endocytosis) and nystatin (caveolae mediated endocytosis). This is with the view to understand the pathway or mechanism whereby the nanoparticles induce cytotoxicity and inflammation. AgNPs were cytotoxic and modulated the inflammatory biomarkers (nitric oxide, interleukin-6, and macrophage migration inhibitory factor) at concentrations ≥ 12.5 µg/ml. However, the inhibitors did not significantly mitigate toxicity nor alter the inflammation caused by the AgNPs. Therefore, for NPs to be utilized to their fullest potential, it is important to evaluate their uptake with respect to toxicity and inflammation, which may also be used to predict any antagonistic cellular responses.