title: In vitro approach to predict post-translational phosphorylation response to mixtures.
abstract: While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited. Realistic exposures potentially consist of hundreds to thousands of chemicals per day, but even relatively simple binary mixture interactions can be inherently difficult to predict based upon the lack of temporal and spatial mechanisms for the individual constituents. To this end, we explore the concept of capitalizing on downstream convergence of intracellular signal transduction to experimentally simplify the means of determining xenobiotics that, when combined, could result in increased or unexpected toxicity. In a proof of principle study, we exposed HepG2 cells to deguelin, a natural isoflavonoid, alone and in combination with KCN, and determined the relative post-translational phosphorylation responses to several key proteins related to mitochondrial outer membrane permeabilization. Dose-dependent phosphorylation activity provides a clear identification of threshold response to low-level exposures, and crosstalk amongst selected proteins correctly forecasts mixtures interactions that may lead to increased toxicity. We then used Bliss Independence to determine if the experimentally measured mixture phosphorylation responses could be predicted with individual responses. Independence accurately predicted mixture interactions for deguelin and KCN (87.5%). To more fully exhaust independence as a model for determining potential pharmacodynamic interactions, we exposed HepG2 cells to deguelin and staurosporine, a broad kinase inhibitor; independence accurately predicted these mixture responses (77.5%). In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of signal transduction and apoptosis.
authors.affiliation: C Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States. Electronic address: jonathan.boyd@mail.wvu.edu.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Boyd
authors.forename: Julie A
authors.initials: JA
authors.lastname: Vrana
authors.forename: Holly N
authors.initials: HN
authors.lastname: Williams
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045424:Complex Mixtures / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity; D056945:Hep G2 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D010539:Permeability; D010766:Phosphorylation; D011190:Potassium Cyanide / Q000633:toxicity; D011237:Predictive Value of Tests; D011499:Protein Processing, Post-Translational; D012402:Rotenone / Q000031:analogs & derivatives / Q000633:toxicity; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods* / Q000592:standards; D015262:Xenobiotics / Q000633:toxicity*
chemical_list: D045424:Complex Mixtures; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D015262:Xenobiotics; D012402:Rotenone; D019311:Staurosporine; C107676:deguelin; D011190:Potassium Cyanide
keywords: Apoptosis; Mixtures; Signal transduction