Combined Effects of High-Dose Bisphenol A and Oxidizing Agent (KBrO3) on Cellular Microenvironment, Gene Expression, and Chromatin Structure of Ku70-deficient Mouse Embryonic Fibroblasts

Abstract

Background: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBrO3).

Objectives: We determined the cellular microenvironment changes induced by co-exposure of BPA and KBrO3 versus either agent alone.

Methods: Ku70-deficient cells were exposed to 150 μM BPA, 20 mM KBrO3, or co-exposed to both agents. Four and 24 hr post-damage initiation by KBrO3, with BPA-only samples timed to coincide with these designated time points, we performed whole-genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH.

Results: We found that 4 hr post-damage initiation, BPA exposure and co-exposure transiently condensed chromatin compared with untreated and KBrO3-only treated cells; the transcription of DNA repair proteins was also reduced. At this time point, BPA exposure and co-exposure also reduced the change in intracellular pH observed after treatment with KBrO3 alone. Twenty-four hours post-damage initiation, BPA-exposed cells showed less condensed chromatin than cells treated with KBrO3 alone; the intracellular pH of the co-exposed cells was significantly reduced compared with untreated and KBrO3-treated cells; and significant up-regulation of DNA repair proteins was observed after co-exposure.

Conclusion: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku70-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the cellular microenvironment that may promote survival.

Citation: Gassman NR, Coskun E, Jaruga P, Dizdaroglu M, Wilson SH. 2016. Combined effects of high-dose bisphenol A and oxidizing agent (KBrO3) on cellular microenvironment, gene expression, and chromatin structure of Ku70-deficient mouse embryonic fibroblasts. Environ Health Perspect 124:1241-1252; http://dx.doi.org/10.1289/EHP237.

Figure 1

Cell survival following co-exposure to BPA and KBrO₃ (from Gassman et al. 2015). Ku70-deficient cells were treated with increasing amounts of KBrO₃ for 1 hr (solid circles) or pre-treated with 150 μM BPA for 1 hr, co-exposed to BPA and increasing amounts of KBrO₃ for 1 hr, and then BPA exposure was continued for a total of 24 hr after KBrO₃ exposure (open circles). After 24 hr, cells were washed, fresh medium was applied, and they were allowed to grow for 6–7 days. Cells were counted, and inhibition of growth was determined as the number of cells remaining after the treatment was compared with the control (% Control).

Figure 2

Gene expression changes observed by whole genome analysis of mRNA isolated 4 hr after treatment with KBrO₃, BPA, or co-exposure to both agents as described in “Methods.” (A) Heat map of gene expression changes observed after treatment was generated using Partek^® Genomic Suite software with probes selected by a fold change cutoff of ± 1.5 compared with untreated controls and an analysis of variance (ANOVA)-calculated significance level of p < 0.01 (n = 3). (B) Significant probe changes identified using the described criteria are sorted by Venn diagram.

Figure 3

Gene expression changes observed by whole genome analysis of mRNA isolated 24 hr after treatment with KBrO₃, BPA, or co-exposure to both agents, as described in “Methods.” (A) Heat map of gene expression changes observed after treatment was generated using Partek^® Genomic Suite software with probes selected by a fold change cutoff of ± 1.5 compared with untreated controls and an analysis of variance (ANOVA)-calculated significance level of p < 0.01 (n = 3). (B) Significant probe changes identified using the described criteria are sorted by Venn diagram.

Figure 4

DNA replication, recombination, and repair networks identified from the uniquely regulated genes identified from the co-exposure condition 24 hr after damage induction by IPA’s Core Analysis module, which searches for enriched canonical pathways. (A) DNA replication, recombination, and repair network 1 (score 46, 31 focus molecules, p-value of top functions 7.18 × 10^–5) is presented with expression values for the co-exposure overlaid, as an indicator of up- or down-regulation (dark gray and white, respectively). (B) DNA replication, recombination, and repair network 3 (score 38, 28 focus molecules, p value of top functions 4.458 × 10^–8) is presented with expression values for the co-exposure overlaid, as an indicator of up- or down-regulation (dark gray and white, respectively).

Figure 5

Levels of chromatin condensation after treatment with KBrO₃, BPA, or co-exposure of both agents at 4 and 24 hr post-damage induction were measured by intensity of Hoechst and PI staining using flow cytometry. (A) Hoechst- and propidium iodide (PI)-stained live cells are sorted by intensity, and the contour maps of the measured intensities for a representative experiment at 4 and 24 hr are shown. Dashed lines show the center of the control contour plot and highlight changes relative to the control cells. (B) Mean intensity values of the Hoechst staining for each treatment condition 4 hr post-damage induction normalized to the control are shown (mean ± SEM of 3 biological replicates). (C) Mean intensities of the Hoechst staining for each treatment condition 24 hr post-damage induction normalized to the control are shown (mean ± SEM). *p < 0.05, with solid and dashed lines showing comparison groups.

Figure 6

Levels of free GSH after treatment with KBrO₃, BPA, or co-exposure to both agents at 4 and 24 hr post-damage induction were measured by staining live cells with ThiolTracker™ Violet and sorting by flow cytometry. (A) ThiolTracker™ Violet live cells were sorted by intensity, and the measured intensities for a representative experiment at 4 and 24 hr are shown. Dashed lines indicate the center of the intensity peak for the control cells and highlight the relative changes in measured intensity compared with the control cells. The long white arrows indicate the direction of decreasing GSH measured by loss of dye intensity. The short white arrow indicates the subpopulation reflecting lower GSH content. (B) Mean intensity values of the ThiolTracker™ Violet staining for each treatment condition 4 hr (black) and 24 hr (red) after damage induction normalized to the control are shown (mean ± SEM of 3 replicates).

Figure 7

Proposed time-line for the cellular changes observed after BPA exposure based on our findings. We hypothesize that DNA repair is inhibited, at both the recognition and excision levels and at the transcription level up to 4 hr after treatment with BPA (in BPA-only cells) or with KBrO₃ (in co-exposed cells), and that between 4 and 24 hr, an adaptive response is induced by BPA co-exposure that results in the up-regulation of DNA repair networks, while alterations in the cellular microenvironment are induced through pH changes and antioxidant depletion. Such changes might result in long-term epigenetic changes or reprogramming events. PTM, post-translational modification.