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Label-free proteomic approach to study the non-lethal effects of silver nanoparticles on a biofilm gut model

ABSTRACT: Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs),due totheir anti-microbial properties, are one of the most commonly utilized. Despite this, the effects of sub-lethal concentrations of AgNPs, especially on gut biofilms, has been poorly investigated. To address these issues, we investigated the proteomic response of a mono-species Escherichia coli gut biofilm, used as in-vitro human gut model, to chronic and acute exposure of sub-lethal concentrations(1 µg/mL) of AgNPs. We used a new gel and label-free proteomic approach based on shotgun nanoflow scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) that, respect to the traditional proteomic investigation, allows a higher dynamic range of quantification of the whole proteome. To assess all different possible exposure scenarios, we compared the total proteome of four samples: untreated cells, cells treated with AgNPs for 24h (acute treatment), for 96h (chronic treatment), and cells grown in the presence of AgNPs for 96h and treated again for 24h (acute and chronic treatment). Proteomic profiling provided new insight into the mechanisms exerted by AgNPs highlighting the important role of the bacterial biofilm in protecting intestinal epithelial cells. Among the 1917 proteins identified, 216 were ANOVA significant and several pathways resulted altered including biofilm formation, bacterial adhesion, ROS stress response and glucose utilization.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Guido Domingo

LAB HEAD: Dr.ssa Milena Marsoni

PROVIDER: PXD014096 | Pride | 2019-10-31

REPOSITORIES: Pride

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Project description:Nanoparticles are compounds of emerging concern with largely unknown risks for human and ecological health. It is crucial to evaluate their potential biological impact to prevent unintended adverse effects on human health and the environment. We analyzed the transcriptional effects of polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) and silver nitrate (AgNO3) on the fathead minnow (Pimephales promelas) to understand their potential toxicity and adverse outcomes. We also tested the feasibility of the fathead minnow as an alternative species to elucidate potential adverse effects on humans. Fathead minnow females were exposed to either 4 Âµg/L of AgNO3 or 70 Âµg/L of PVP-AgNPs for 96h. Microarray analyses were performed on liver and brain. Functional analysis identified potential toxicity pathways and molecular initiating events (MIEs) that were confirmed with functional assays. Data suggested that AgNO3 and PVP-AgNPs had both common and distinct transcriptional effects. The nanoparticles were linked to neurotoxicity and oxidative stress, and identified as a dopamine receptor antagonist. Silver nitrate was also identified as a potential neurotoxicant and was confirmed as adrenergic and cannabinoid receptors antagonist. While silver nitrate and PVP-AgNPs were both potential neurotoxicants, they appeared to act through different MIEs. Fathead minnow is a promising alternative species to elucidate potential adverse effects of relevance to human health. We analyzed the transcriptional effects of polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) and silver nitrate (AgNO3) on the fathead minnow (Pimephales promelas) to understand their potential toxicity and adverse outcomes. FHM were obtained from Aquatic Biosystems (Fort Collins, CO), held in aerated dechlorinated tap water and fed three times daily with ZeiglerÂ® AquaTox Feed Gardners, PA, USA). Fathead minnow females were exposed to either 4 Âµg/L of AgNO3 or 70 Âµg/L of PVP-AgNPs (Luna Innovations, Blackburn, VA) for 96h at 24Â°C Â± 1 with a 90% water change at 48 hours. Microarray analyses were performed on liver and brain.

Project description:The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3 were exposed for 6h and 24h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics, and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism. In total 73 samples are analyzed (24 different samples all n=3; except for 1 sample with n=4). Twelve of the 24 different samples are extracted from Caco-2 cells, 6 samples at t=6h and 6 at t=24h The other 12 different of the 24 samples are extracted from MCF-7 cells, 6 samples at t=6h and 6 at t=24h For each cell type and each timepoint one of the 6 samples was a negative control sample

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	20160617_28_C_3.raw	Raw
	20160617_30_C_NSP_1.raw	Raw

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Label-free proteomic approach to study the non-lethal effects of silver nanoparticles on a biofilm gut model

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