Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening

Abstract

A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.

Figure 1

Complete screening dataset. Dose response curves for the FCCP control titrations and 1,408 compounds tested in the primary screening for 1 (left) and 5 (right) h (Red, FCCP positive control; blue, ratio (red/green, 590 nm/535 nm) curve classes 1.1, 1.2 or 2.1; green ratio(red/green) curve classes 1.3, 1.4, 2.2, 2.3, 2.4, and 3, and gray, ratio(red/green) curve class 4).^,

Figure 2

Several flavonoids that decreased mitochondrial membrane potential were identified in the primary screening. A) Changes in mitochondrial membrane potential in intact HepG2 cells generated by three flavonoids identified. (Left, 1 h; right, 5 h). B) A representative image of HepG2 cells stained with Mito-MPS dye in the absence or in the presence of kaempferol. Images acquired in ImageXpress microsystem using a 20× objective. While red fluorescent aggregates are localized in the mitochondria, green fluorescent monomers are mainly in the cytosol. The composed images are the merger of red, blue (nuclei), and green fluorescence.

Figure 2

Several flavonoids that decreased mitochondrial membrane potential were identified in the primary screening. A) Changes in mitochondrial membrane potential in intact HepG2 cells generated by three flavonoids identified. (Left, 1 h; right, 5 h). B) A representative image of HepG2 cells stained with Mito-MPS dye in the absence or in the presence of kaempferol. Images acquired in ImageXpress microsystem using a 20× objective. While red fluorescent aggregates are localized in the mitochondria, green fluorescent monomers are mainly in the cytosol. The composed images are the merger of red, blue (nuclei), and green fluorescence.