Oxidative stress is known to play important roles in engineered nanomaterial-induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial-mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS-2B and nanoscale titanium dioxide. Utilizing 2-DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2-DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses™ canonical pathways and Ingenuity Pathway Analyses tox lists to create protein-interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS-2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide-treated BEAS-2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein-interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.

Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based invitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking invitro-based measurements to chemical effects on adverse human health outcomes.

Recently, investigators demonstrated associations between fine particulate matter (PM)-associated metals and adverse health effects. Residual oil fly ash (ROFA), a waste product of fossil fuel combustion from boilers, is rich in the transition metals Fe, Ni, and V, and when released as a fugitive particle, is an important contributor to ambient fine particulate air pollution. We hypothesized that a single-inhalation exposure to transition metal-rich PM will cause concentration-dependent cardiovascular toxicity in spontaneously hypertensive (SH) rats. Rats implanted with telemeters to monitor heart rate and electrocardiogram were exposed once by nose-only inhalation for 4 hours to 3.5 mg/m(3), 1.0 mg/m(3), or 0.45 mg/m(3) of a synthetic PM (dried salt solution), similar in composition to a well-studied ROFA sample consisting of Fe, Ni, and V. Exposure to the highest concentration of PM decreased T-wave amplitude and area, caused ST depression, reduced heart rate (HR), and increased nonconducted P-wave arrhythmias. These changes were accompanied by increased pulmonary inflammation, lung resistance, and vagal tone, as indicated by changes in markers of HR variability (increased root of the mean of squared differences of adjacent RR intervals [RMSSD], low frequency [LF], high frequency [HF], and decreased LF/HF), and attenuated myocardial micro-RNA (RNA segments that suppress translation by targeting messenger RNA) expression. The low and intermediate concentrations of PM had less effect on the inflammatory, HR variability, and micro-RNA endpoints, but still caused significant reductions in HR. In addition, the intermediate concentration caused ST depression and increased QRS area, whereas the low concentration increased the T-wave parameters. Thus, PM-induced cardiac dysfunction is mediated by multiple mechanisms that may be dependent on PM concentration and myocardial vulnerability (this abstract does not reflect the policy of the United States Environmental Protection Agency).

Changes in the serum proteome were identified during early, fulminant, and recovery phases of liver injury from acetaminophen in the rat. Male F344 rats received a single, noninjury dose or a high, injury-producing dose of acetaminophen for evaluation at 6 to 120 h. Two-dimensional gel electrophoresis of immunodepleted serum separated approximately 800 stained proteins per sample from which differentially expressed proteins were identified by mass spectrometry. Serum alanine aminotransferase/aspartate aminotransferase levels and histopathology revealed the greatest liver damage at 24 and 48 h after high-dose acetaminophen corresponding to the time of greatest serum protein alterations. After 24 h, 68 serum proteins were significantly altered of which 23 proteins were increased by Ͼ5-fold and 20 proteins were newly present compared with controls. Only minimal changes in serum proteins were noted at the low dose without any histopathology. Of the 54 total protein isoforms identified by mass spectrometry, gene ontology processes for 38 unique serum proteins revealed involvement of acute phase response, coagulation, protein degradation, intermediary metabolism, and various carrier proteins. Elevated serum tumor necrosis factor-␣ from 24 to 48 h suggested a mild inflammatory response accompanied by increased antioxidant capability demonstrated by increased serum catalase activity. Antibody array and enzyme-linked immunosorbent assay analyses also showed elevation in the chemokine monocyte chemoattractant protein-1 and the metalloprotease inhibitor tissue inhibitor of metalloproteinases-1 during this same period of liver injury. This study demonstrates that serum proteome alterations probably reflect both liver damage and a concerted, complex response of the body for organ repair and recovery during acute hepatic injury.