Academic journal article Environmental Health Perspectives

Using ToxCast[TM] Data to Reconstruct Dynamic Cell State Trajectories and Estimate Toxicological Points of Departure

Academic journal article Environmental Health Perspectives

Using ToxCast[TM] Data to Reconstruct Dynamic Cell State Trajectories and Estimate Toxicological Points of Departure

Article excerpt


A major focus in public health has been to understand and limit potential adverse health effects of chemicals. However, despite an expectation of safety by the general public, there are tens of thousands of chemicals in commerce that have been evaluated on the basis of closely related analogs but that lack chemical-specific toxicity information (Judson et al. 2009). This lack of toxicity information has led to national and international efforts to use in vitro high-throughput screening (HTS) methods to collect data on biochemical and cellular responses following chemical treatment in vitro (Kavlock et al. 2009; Attene-Ramos et al. 2013). A key element of toxicity testing in the 21st century [National Research Council Committee on Toxicity Testing and Assessment of Environmental Agents (NRC) 2007; Boekelheide and Andersen 2010] is conceptually organizing HTS data into pathways that, when sufficiently perturbed, lead to adverse outcomes. One challenge associated with this new vision has been the assessment of "tipping points" beyond which pathway perturbations invoke a lasting change that could ultimately lead to an adverse effect.

The present study is part of the U.S. Environmental Protection Agency's (EPA's) ToxCast[TM] project, which aims to develop in vitro screens to identify potentially hazardous substances for further targeted testing (Kavlock et al. 2012). We used high-content imaging (HCI) (Giuliano et al. 2006), which applies automated image analysis techniques to capture multiple cytological features using fluorescent labels, to measure the concentration-dependent dynamic changes in the state of HepG2 cells. Although they are not fully metabolically capable, HepG2 cells can undergo continuous proliferation in culture and have a demonstrated capacity to predict hepatotoxicity of pharmaceutical compounds with good sensitivity and specificity (O'Brien et al. 2006; Abraham et al. 2008). We used computational tools to deconvolute HCI responses into cell-state trajectories and to analyze them for their propensity to recover to normal (basal) conditions over the test period. The critical concentrations associated with nonrecoverable cellular trajectories were determined, where possible, and compiled into a novel chemical classification scheme. We discuss how these "tipping points" in the function of cellular systems might be used to define a point of departure for risk-based prioritization of environmental chemicals.


Cell Culture

HepG2 cells were obtained from American Type Culture Collection (ATCC) and used before passage 20. Cells were maintained and expanded in complete media [10% fetal bovine serum (FBS) in Minimum Essential Medium with Earle's Balanced Salt Solution (MEM/EBSS) supplemented with penicillin/streptomycin, L-glutamine, and nonessential amino acids]. Cell culture reagents were obtained from VWR International. HepG2 cells were harvested by trypsinization and plated at different densities in 25 [micro]L of culture medium, depending on incubation time, in clear-bottom, 384-well microplates (Falcon #3962) that were coated with rat tail collagen I. The cells were incubated overnight to allow attachment and spreading.

Chemical Treatments

HepG2 cells were treated with 967 chemicals from ToxCast[TM] Phase I and Phase II libraries (U.S. EPA 2014). Cells were treated with dimethyl sulfoxide (DMSO) as a solvent control at a final concentration of 0.5% v/v or with compounds in DMSO with a resulting final DMSO concentration of 0.5% v/v. Compound treatment was done at concentrations of 0.39, 0.78, 1.56, 3.12, 6.24, 12.5, 25, 50, 100, and 200 [micro]M in duplicate on each plate. Cells were treated with ToxCast[TM] Phase I compounds for 1, 24, and 72 hr and ToxCast[TM] Phase II compounds for 24 and 72 hr only. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and taxol were used as positive controls for mitochondrial function and cytoskeletal stability, respectively; DMSO served as the negative control for this experiment. …

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