PCB11 Metabolite, 3,3'-Dichlorobiphenyl-4-ol, Exposure Alters the Expression of Genes Governing Fatty Acid Metabolism in the Absence of Functional Sirtuin 3: Examining the Contribution of MnSOD

Abstract

Although the production of polychlorinated biphenyls (PCBs) is prohibited, the inadvertent production of certain lower-chlorinated PCB congeners still threatens human health. We and others have identified 3,3'-dichlorobiphenyl (PCB11) and its metabolite, 3,3'-dichlorobiphenyl-4-ol (4OH-PCB11), in human blood, and there is a correlation between exposure to this metabolite and mitochondrial oxidative stress in mammalian cells. Here, we evaluated the downstream effects of 4OH-PCB11 on mitochondrial metabolism and function in the presence and absence of functional Sirtuin 3 (SIRT3), a mitochondrial fidelity protein that protects redox homeostasis. A 24 h exposure to 3 μM 4OH-PCB11 significantly decreased the cellular growth and mitochondrial membrane potential of SIRT3-knockout mouse embryonic fibroblasts (MEFs). Only wild-type cells demonstrated an increase in Manganese superoxide dismutase (MnSOD) activity in response to 4OH-PCB11⁻induced oxidative injury. This suggests the presence of a SIRT3-mediated post-translational modification to MnSOD, which was impaired in SIRT3-knockout MEFs, which counters the PCB insult. We found that 4OH-PCB11 increased mitochondrial respiration and endogenous fatty-acid oxidation-associated oxygen consumption in SIRT3-knockout MEFs; this appeared to occur because the cells exhausted their reserve respiratory capacity. To determine whether these changes in mitochondrial respiration were accompanied by similar changes in the regulation of fatty acid metabolism, we performed quantitative real-time polymerase chain reaction (qRT-PCR) after a 24 h treatment with 4OH-PCB11. In SIRT3-knockout MEFs, 4OH-PCB11 significantly increased the expression of ten genes controlling fatty acid biosynthesis, metabolism, and transport. When we overexpressed MnSOD in these cells, the expression of six of these genes returned to the baseline level, suggesting that the protective role of SIRT3 against 4OH-PCB11 is partially governed by MnSOD activity.

Keywords: 4OH-PCB11; MnSOD; PCB 11; Sirtuin 3; fatty acid metabolism; mitochondria.

Figure 1

Treatment with 3 μM 4OH-PCB11 for 24 h decreased the cell growth in SIRT3-KO MEFs. (A) Doubling times of WT and SIRT3-KO MEFs treated with Vehicle (0.1% v/v DMSO) or 3 μM 4OH-PCB11 for 24 h. Data are mean doubling times of cells from six treatment dishes from two separate experiments performed in triplicate (± 1SD), n = 6. # p < 0.05, as compared to the vehicle-treated SIRT3-KO group. (B) Western blot analysis of Sirt3 immunoreactive protein in WT and SIRT3-KO MEFs following 24 h treatment with 3 μM 4OH-PCB11 or vehicle. β-actin was used as a loading control. The quantitation was presented as the average + 1SD ratio of SIRT3 to β-actin in fluorescence units from three different blots * p < 0.05 as compared to vehicle-treated SIRT3-WT group.

Figure 2

Steady-state levels of reactive oxygen species (ROS) and mitochondrial membrane potential were determined by Mitosox oxidation. (A) Asynchronous cultures of MEFs were incubated with 3 μM 4OH-PCB11 for 24 h. Cells were trypsinized, washed once with PBS, and labeled with 2 μM Mitosox (in 0.1% DMSO, 20 min) in PBS containing 5 mM pyruvate at 37 °C. (B) Mitochondrial membrane potential as measured by JC-1 (5 μg/mL, 15 min). The mean fluorescence intensity (MFI) of 10,000 cells was analyzed by flow cytometry. Samples were assayed in triplicate; data are the means ± 1SEM of two independent experiments, each containing three treatment dishes, n = 6. * p < 0.05 as compared to vehicle-treated WT control group. ## p < 0.01 as compared to vehicle-treated SIRT3-KO control group.

Figure 3

MnSOD activity was induced only in 4OH-PCB11-treated WT MEFs. (A) Asynchronous cultures of WT and SIRT3-KO MEFs were incubated with 3 μM 4OH-PCB11 for 24 h. Cells were harvested, scraped, and pelleted in PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer for western blot. Quantitation was presented as the average ratio of MnSOD to β-actin in fluorescence units obtained from two different blots (B) To measure MnSOD activity, whole cell homogenates were prepared from the pellets and resuspended in 50 mM potassium phosphate buffer (pH 7.8) for spectrophotometric analysis. Data were normalized from two independent experiments. Data represent the mean of each group ± 1SEM (n = 6). ** p < 0.01 as compared to vehicle-treated WT control group.

Figure 4

Basal and ATP-linked respiration increased in SIRT3-KO MEFs, and reserve respiratory capacity decreased. WT and SIRT3-KO MEFs were trypsinized and plated in Seahorse XF96 cell culture plates. The next day, cells were treated with 3 μM 4OH-PCB11 or vehicle for an additional 24 h. Cells were washed in buffered DMEM and then changed to assay medium. The oxygen consumption rate (OCR) was measured with sequential injections of oligomycin, FCCP, and antimycin A-rotenone mixture (upper panel). The OCR was calculated and plotted for basal, ATP-linked, proton leak, reserved respiratory, and non-mitochondrial respiration. Data shown are representative of three independent experiments. Data are the mean ± 1SEM (n = 18–22). ** p < 0.01; **** p < 0.0001 as compared to vehicle-treated WT control group. ## p < 0.01 and #### p < 0.0001 as compared to vehicle-treated SIRT3-KO control group.

Figure 5

Endogenous fatty acid oxidation (FAO)-driven respiration increased only in SIRT3-KO MEFs following 4OH-PCB11 treatment. WT and SIRT3-KO MEFs were trypsinized, and 15,000 cells were plated in Seahorse XF96 cell culture plates. After 24 h, cells were treated with 3 μM 4OH-PCB11 or vehicle for an additional 24 h. At 8 h before metabolic flux analysis, the culture medium was replaced with substrate-limited medium, and 45 min before measuring the oxygen consumption rate, the medium was replaced with FAO assay medium. Immediately before placing the plates into the XF analyzer, the wells received BSA or palmitate-BSA substrate to distinguish between endogenous (A) and exogenous (B) FAO-driven respiration. After the baseline OCR was established, etomoxir (40 μM) was injected to determine the amount of FAO-associated OCR (calculated as the difference in OCR before and after etomoxir injection). Data are representative of two independent experiments. Data are the mean ± 1SEM (n = 18–22). * p < 0.05; ** p < 0.01; **** p < 0.0001 as compared to vehicle-treated WT control group. # p < 0.05 and ### p < 0.001 as compared to vehicle-treated SIRT3-KO control group.

Figure 6

4OH-PCB11 caused increased expression of genes associated with fatty acid oxidation in SIRT3-KO MEFs. WT and SIRT3-KO MEFs were trypsinized and plated. After 24 h, cells were treated in triplicate with 0.1, 1, and 3 μM of 4OH-PCB11 for an additional 24 h. To quantitate mRNA expression for fatty acid metabolism, total RNA was extracted with a QIAGEN RNA Extraction kit. Total RNA was reverse transcribed, and qRT-PCR was performed. The CT values for the target genes were normalized to the 18S rRNA transcript, the fold difference (relative abundance) was calculated using the formula 2-ΔΔCT, and the mean was plotted. N = 3; * p < 0.05; ** p < 0.01.

Figure 7

Overexpression of MnSOD could reverse the 4OH-PCB11 induced increases in expression of genes associated with FAO as well as decrease the elevated mitochondrial respiration following polychlorinated biphenyl (PCB) treatment. SIRT3-KO MEFs were incubated with replication-incompetent adenoviral vectors (100 MOI), AdCMV Bgl II (AdBgl II), or AdCMV MnSOD (AdMnSOD) for 24 h. Then, the medium was changed, and the cells were incubated for an additional day before treatment with 3 μM 4OH-PCB11 or vehicle for 24 h. qRT-PCR was repeated for the FAO pathway genes that were significantly upregulated in the previous experiment, see Figure 6 (n = 3). * p < 0.05 compared to AdBgl II control (A). MnSOD activity was measured in cell homogenates. Data represents the average and error range of two independent treatment dishes per group (B) The oxygen consumption rate (OCR) was measured with Seahorse Extracellular Flux analyzer. Data are the mean ± 1SEM of independently infected and treated 14–16 wells. *** p < 0.001 as compared to vehicle-treated AdBgl II group. ## p < 0.01 and ### p < 0.001 as compared to vehicle-treated AdMnSOD group (C).