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In Vitro Exposures of A549, J774A.1 Cells to SiO2, TiO2 Nanoforms and Related Cellular and Molecular Level Effects: Application of Proteomics

ABSTRACT: There is emerging interest in using proteomic data for environmental health-risk assessments. Meanwhile, due to attractive physicochemical properties, production and use of engineered nanomaterials (ENMs) are expanding with potential for exposure, thus necessitating toxicity information on these materials for human health risk analysis, where proteomic data can be informative. Here, cells (A549 human lung epithelial, J774 mouse monocyte/macrophage) were exposed to ENMs (nanoforms of SiO2, TiO2) of different sizes, surface chemistries (dose:0-100 ug/cm2, 24 h) for in vitro toxicity data. Cellular cytotoxicity (CTB, ATP, LDH), oxidative stress and proteomic analysis (MS-, antibody-based) were conducted post-nanoparticle (NP) exposures to determine cytotoxic potencies and identify molecular-level effects. Dose-, nanoform-, cell type-specific cytotoxicity changes were seen with both nanoSiO2 and nanoTiO2 exposures. Size, agglomeration, surface groups and metal impurities appeared to be cytotoxicity determinants. Proteomic data identified some common enriched mechanistic pathways relevant to phagocytosis, cell-shape changes, apoptosis, and inflammatory processes for nanoSiO2, nanoTiO2 exposed J774 cells. A549 cells exhibited pathway changes relevant to cell metabolism, endocytosis and inflammatory processes post-NP exposures. Concordance was seen between, cytotoxicity responses, notably cellular ATP, critical for cell viability, cellular oxidative stress and affected cellular pathways. These findings demonstrate the utility of proteomics in toxicology, warranting further exploration.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Dr. Premkumari Kumarathasan

PROVIDER: MSV000095579 | MassIVE | Mon Aug 12 11:57:00 BST 2024

REPOSITORIES: MassIVE

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Project description:Pulmonary exposure to high doses of nanoparticles (NP) leads to well characterized lung toxicity in addition to long-term NP retention. However, pulmonary NP accumulation and toxicity following low dose exposures are not well described. In the present study we sought to: (1) investigate particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO2) and (2) determine the effects of long-term particle accumulation on pulmonary systems. Female C57BL/6 mice were exposed to rutile nano-TiO2 (primary size of 20.6 nm and surface area of 107.7 m2/g) via single intratracheal instillations of 18, 54 and 162 M-BM-5g/mouse and sampled 1, 3 and 28 days post-exposure. The deposition of nano-TiO2 in the lungs was assessed using Nanoscale Hyperspectral Microscope. DNA microarrays, pathway-specific real-time RT-PCR (qPCR) and gene-specific qPCR arrays, and tissue protein analyses were employed to characterize pulmonary responses. Hyperspectral mapping showed dose-dependent retention of nano-TiO2 in the lungs up to 28 days post-exposure time. Retention did not correlate with the extent of inflammatory neutrophil influx into the lungs. DNA microarray analysis showed altered expression of approximately 3000 genes across all treatment groups (M-BM-11.3 fold; p<0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein levels. Although the low dose exposure failed to induce observable inflammation, significant changes in the expression of genes and proteins associated with inflammation were observed. Moreover, diminished (or absent) neutrophil influx in the low and medium dose groups was correlated with negative regulation of genes associated with ion homeostasis and muscle regulation. Gene expression changes for several inflammatory mediators have previously been noted in mice exposed to the same nano-TiO2 via inhalation. Our results suggest that retention of nano-TiO2 in the absence of inflammation and effective clearance can perturb calcium and ion homeostasis, and affect smooth muscle activities over time. This experiment consists of three different dosages of TiO2, e.g., low (18 ug), medium (54 ug) and high (162 ug), and one control. There are 3 time points for each treatment and control group, e.g., day 1, day 3 and day 28. Each dose or time point has 5-6 biological replicates. There are total 65 samples (arrays)

Project description:Pulmonary exposure to high doses of nanoparticles (NP) leads to well characterized lung toxicity in addition to long-term NP retention. However, pulmonary NP accumulation and toxicity following low dose exposures are not well described. In the present study we sought to: (1) investigate particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO2) and (2) determine the effects of long-term particle accumulation on pulmonary systems. Female C57BL/6 mice were exposed to rutile nano-TiO2 (primary size of 20.6 nm and surface area of 107.7 m2/g) via single intratracheal instillations of 18, 54 and 162 µg/mouse and sampled 1, 3 and 28 days post-exposure. The deposition of nano-TiO2 in the lungs was assessed using Nanoscale Hyperspectral Microscope. DNA microarrays, pathway-specific real-time RT-PCR (qPCR) and gene-specific qPCR arrays, and tissue protein analyses were employed to characterize pulmonary responses. Hyperspectral mapping showed dose-dependent retention of nano-TiO2 in the lungs up to 28 days post-exposure time. Retention did not correlate with the extent of inflammatory neutrophil influx into the lungs. DNA microarray analysis showed altered expression of approximately 3000 genes across all treatment groups (±1.3 fold; p<0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein levels. Although the low dose exposure failed to induce observable inflammation, significant changes in the expression of genes and proteins associated with inflammation were observed. Moreover, diminished (or absent) neutrophil influx in the low and medium dose groups was correlated with negative regulation of genes associated with ion homeostasis and muscle regulation. Gene expression changes for several inflammatory mediators have previously been noted in mice exposed to the same nano-TiO2 via inhalation. Our results suggest that retention of nano-TiO2 in the absence of inflammation and effective clearance can perturb calcium and ion homeostasis, and affect smooth muscle activities over time.

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In Vitro Exposures of A549, J774A.1 Cells to SiO2, TiO2 Nanoforms and Related Cellular and Molecular Level Effects: Application of Proteomics

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