DOI: 10.25820/data.007101
title: Dataset for Astrocyte Mitochondria are a Sensitive Target of PCB52 and its Human-Relevant Metabolites

description: The C6 cells, a rat astroglial cell line was exposed to previously identified IC50 concentrations of PCB52 and its human-relevant metabolites, 4-OH-PCB52 and 4-OH-PCB52 sulfate. Various endpoint assays were performed to assess mitochondria as cellular targets of PCB52 and its human-relevant metabolites. The Glucose-galactose assay indicates that mitochondria may be the target of the three test compounds. Further, the DCFDA assay shows generation of oxidative stress and a fluorescence based assay for oxidative stress shows increased oxidative stress being generated in mitochondria of C6 cells exposed to PCB52 metabolites. JC-10 assay also shows loss of mitochondrial membrane potential, with 4-OH-PCB52 being the most potent compound. In addition, immunofluorescence assay for HSP60, a mitochondrial matrix protein, followed by confocal imaging show that mitochondrial morphology is altered distinctly by PCB52 and its human-relevant metabolites, 4-OH-PCB52 and 4-OH-PCB52 sulfate. Further, the seahorse assay assessing oxygen consumption rate in C6 cells is altered upon exposure to PCB52, 4-OH-PCB52, and 4-OH-PCB52 sulfate.
description: PCBs used: PCB52, 4-OH-PCB52 and 4-OH-PCB52 sulfate were synthesized and provided by the Synthesis Core, Iowa Superfund Research Program. Previosly identified IC50 concentrations (https://doi.org/10.1021/acs.chemrestox.3c00095) were used for all the assays except for the Glucose-Galactose assay. All exposures were in serum-free, phenol-red free cell culture media.

Glucose-Galactose assay: C6 cells were exposed to varying concentrations (0 - 20 micromoles) of PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate for 24 hours, after which alamar blue assay was performed to assess cell viability of C6 cells.

Assessment of oxidative stress: Generation of oxidative stress was assessed by two methods: DCFDA assay and live cell imaging of C6 cells stained with MitoSox Red or CellRox Green, fluorescent probes used for detection of mitochondrial and cellular oxidative stress. Both the assays were performed after 2 hour exposure. Images were analyzed using FIJI (ImageJ), an open-source image analysis software using a macro available at: https://github.com/ststrack/Strack-Lab-software

Mitochondrial membrane potential: C6 cells were exposed to PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate for 2 hours after which relative mitochondrial membrane potential was assessed using Abcam JC10 assay. The fluorescence for red aggregate form and green monomeric form has been reported. A ratio of green monomeric form and red aggregate form was used to calculate relative membrane potential.

Mitochondrial structure assessment: C6 cells exposed to PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate for 2 h were fixed and immunofluorescence staining was performed for HSP60, a mitochondrial matrix protein. Hoechst 33342 was used to stain cell nuclei. Confocal images were taken on Zeiss LSM 980 microscope. Image analysis was done using FIJI (ImageJ), an open-source image analysis software using a macro available at: https://github.com/ststrack/Strack-Lab-software

Seahorse Assay: C6 cells were exposed to PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate for 2 h after which Seahorse assay was performed to assess oxygen consumption rate in C6 cells, as a measure of mitochondrial function.

MitoQ/MitoTempo assay: C6 cells were pretreated with varying concentrations of MitoQ or MitoTempo, two mitochondria targeted antioxidants for 2 h. After this, cells were washed and exposed to PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate at IC50 concentrations and alamar blue assay was performed to assess cell viability of C6 cells, 24 hours post exposure.

Caspase 3/7 Glo assay: C6 cells were exposed to PCB52 or 4-OH-PCB52 or 4-OH-PCB52 sulfate for varying time duration. Promega caspase 3/7 glo assay, a luminescence based assay was used to assess induction of caspases 3 and 7 upon exposure to the test compounds.