Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures

Abstract

In this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.

Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis

“…Changes in the proteome of A549 cells after 24 h of exposure to CR and MI particles were assessed via 2D‐GE as described previously (Vuong et al ., ,b). The results presented in Supporting information Table S1 revealed that the expressions of 49 protein spots were significantly affected by both CR and MI (two‐way ANOVA: Treatment × Dose interaction, Treatment or Dose main effects, P < 0.05).…”

“…Total protein from the A549 cells (control and particle‐exposed) was extracted and examined by two‐dimensional gel electrophoresis (2D‐GE) as previously described (Vuong et al ., ,b). Following electrophoresis, the gel was washed for 30 min in water, stained in BioSafeCoomassie Blue (Bio‐Rad) overnight (16–20 h), destained twice in water (20 min) and then imaged with a standard scanner.…”

“…To overcome the typical warping and distortion issues from gel to gel particularly near the extremities of the pH range and the molecular weight, a common area across all experimental gels that clearly shows the protein spots was selected to assess the proteome differences among the treatments, where proteins in the window of pH 5.1–7.8 and 100–20 kDa were analyzed (Vuong et al ., ,b). A total of 543 well‐resolved protein spots in this common area were compared across all experimental gels, and the identities of 333 of these protein spots were determined via matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry (MALDI‐TOF‐TOF‐MS; Vuong et al ., ,b). The protein spots within the gels were matched and quantified with PDQuestTM Advance V8.0.1 (Bio‐Rad), where spot volume was quantified using the available “Local regression model (LOESS)” algorithm in PDQuest.…”

“…Thereby, it is fundamental to assess whether different forms of silica can trigger differential responses at the cellular level. Recently, we demonstrated that toxicoproteomics is a useful approach to identify and differentiate the mechanisms of particle toxicity of two respirable particles that are physically and chemically different such as titanium dioxide and carbon black (Vuong et al, 2016a). In this study, we questioned whether in vitro toxicoproteomics in conjunction with gene expression analysis were sensitive enough to differentiate the effect of two particles that are identical in chemical formula (silicon dioxide) but differed only in their physical properties.…”

In vitro toxicoproteomic analysis of A549 human lung epithelial cells exposed to urban air particulate matter and its water-soluble and insoluble fractions

“…However, a detailed proteomic investigation to assess the molecular mechanisms delineating the toxic effects of EHC-93 as a whole (total) or separated fractions (i.e., insoluble and soluble) has not been conducted. In our recent studies, we demonstrated that in vitro toxicoproteomics is an approach that is capable of distinguishing the pathways associated with cytotoxic effects of respirable particles that are different in physicochemical properties such as carbon black and titanium dioxide [ 62 , 63 ]. Furthermore, we also showed that our in vitro toxicoproteomic approach was capable of differentiating the effects of particles that were identical in chemical formula (SiO 2 ) but differed in physical properties such as cristobalite and α-quartz [ 61 ].…”