An Approach to Investigate Intracellular Protein Network Responses

Abstract

Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common endpoint evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations amongst three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress, and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, amongst these 3 traditionally linear MAPK cascades was further probed by co-exposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems.

Application of human cell transformation assay on assessment of carcinogenic potential of river organic pollutants

“…17 Human cell-based in vitro systems can be applied for testing the biological effects of single toxicants or environmental mixtures, and for the evaluation of environmental quality in time course studies. 17 Human cell-based in vitro systems can be applied for testing the biological effects of single toxicants or environmental mixtures, and for the evaluation of environmental quality in time course studies.…”

“…The approach of combining multiple toxicological endpoints with a system-based impact assessment allows us to obtain an insight into time-and dose-dependent molecular perturbations of specific biological pathways. 17 Human cell-based in vitro systems can be applied for testing the biological effects of single toxicants or environmental mixtures, and for the evaluation of environmental quality in time course studies. In this study, organic extracts from the surface water and shallow groundwater of the Jialu River basin were subjected to in vitro bioassays.…”

Biological network analysis with CentiScaPe: centralities and experimental dataset integration

“…An example of usage of weighted networks centralities analysis, can be found in Holly et al 9 where an euclidean distance is given to each edge depending on the difference between the phosphorylation level of the proteins connected by that edge.…”

Characterization of the ERK1/2 phosphorylation profile in human and fish liver cells upon exposure to chemicals of environmental concern

“…Indeed, it has been proposed that perturbations of the signaling network may serve as an excellent forecaster of cell death that occur days or weeks after the perturbation has occurred (Vrana et al, 2014). This feature makes cell signaling pathways excellent indicators of exposure identification and hazard prediction (Currie et al, 2014). Secondly, using permanent cell lines provides a mean to test a wide range of concentrations and exposure times while zooming in on a specific response that is measured directly.…”