Respirometric Screening and Characterization of Mitochondrial Toxicants Within the ToxCast Phase I and II Chemical Libraries.

Mitochondrial toxicity drives several adverse health outcomes. Current high-throughput screening assays for chemically induced mitochondrial toxicity typically measure changes to mitochondrial structure and may not detect known mitochondrial toxicants. We adapted a respirometric screening assay (RSA) measuring mitochondrial function to screen ToxCast chemicals in HepG2 cells using a tiered testing strategy. Of 1042 chemicals initially screened at a singlemaximal concentration, 243 actives were identified and rescreened at 7 concentrations. Concentration-response data for 3 respiration phases confirmed activity and indicated a mechanism for 193 mitochondrial toxicants: 149 electron transport chain inhibitors (ETCi), 15 uncouplers and 29 adenosine triphosphate synthase inhibitors. Subsequently, an electron flow assay was used to identify the target complex for 84 of the 149 ETCi. Sixty reference chemicals were used to compare the RSA to existing ToxCast and Tox21 mitochondrial toxicity assays. The RSA was most predictive (accuracy = 90%) of mitochondrial toxicity. The Tox21 mitochondrial membrane potential assay was also highly predictive (accuracy = 87%) of bioactivity but underestimated the potency of well-known ETCi and provided no mechanistic information. The tiered RSA approach accurately identifies and characterizes mitochondrial toxicants acting through diverse mechanisms and at a throughput sufficient to screen large chemical inventories. The electron flow assay provides additional confirmation and detailed mechanistic understanding for ETCi, the most common type of mitochondrial toxicants among ToxCast chemicals. The mitochondrial toxicity screening approach described herein may inform hazard assessment and the in vitro bioactive concentrations used to derive relevant doses for screening level chemical assessment using new approach methodologies.

Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: effect of size, surface coating, and intracellular uptake.

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO3), and CeO2 nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO3 exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO3. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO3 evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO3. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO3, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.