Polychlorinated biphenyls (PCBs) are synthetic organic chemicals consisting of a biphenyl and various numbers of chlorine atoms. For more than 50 years, PCBs were produced commercially (Aroclors) and marketed for various industrial applications, including use in capacitors, transformers, plasticizers, surface coatings, inks, adhesives, and pesticides—until their adverse effects on health became evident [1
]. Chronic exposure to mixtures of PCBs is associated with a wide range of toxic effects, including hepatotoxicity, carcinogenicity, hormonal disruption, and neurotoxicity [2
]. Although the production of PCBs was banned in North America in 1977, and PCBs are no longer used in manufacturing, a unique PCB congener (3,3′-dichlorobiphenyl [PCB11]) was detected in soil and air samples from New York, Chicago, and Philadelphia; notably, this congener was not originally a component of Aroclors [3
]. The same group also identified PCB11 and other PCB congeners (due to unintended production during yellow pigment manufacturing) in commercial paints, inks, textiles, paper, cosmetics, leather, plastics, and food. Thus, the general population is exposed on a daily basis to PCBs [5
]. Furthermore, Marek et al., as well as Sethi et al., detected PCB11 in human samples [6
], and our group reported measurable levels of hydroxylated PCB11 metabolites, including 4OH-PCB11, in human blood [8
]. Thus, understanding the PCB-induced health effects at a molecular level could help establish strategies to overcome the health effects of PCB exposure.
Studies from the past ten years strongly suggest that PCBs and PCB metabolites, such as 4-chlorobenzoquinone and 4OH-PCB11, induce mitochondrial reactive oxygen species (ROS) and oxidative stress in cultured cells and mice [8
]. In Zhu et al. (2009), we showed that exposure to PCBs and PCB metabolites increased the activity of MnSOD, the major mitochondrial enzyme responsible for removing O2
]. Interestingly, this increase in MnSOD activity was not accompanied by an increase in MnSOD immunoreactive protein, suggesting that MnSOD was regulated post-translationally. Further, SIRT3, as the major deacetylase in mitochondria, is capable of affecting MnSOD by deacetylating its critical lysines to regulate its activity [13
]. SIRT3 appears to decrease in abundance during aging as a result of a high fat-diet or metal toxicity, and in metabolic disease states such as type II diabetes [16
]. Because SIRT3 regulates mitochondrial metabolism during stress, it has the potential to be an important player in PCB-induced changes to mitochondrial function and metabolism.
Here, we conducted a series of experiments to determine the effects of 4OH-PCB11 on mitochondrial function and metabolism in the presence and absence of functional SIRT3 in vitro. By overexpressing MnSOD, we also investigated the role of this mitochondrial enzyme in the 4OH-PCB11-induced metabolic changes.
Cellular injury and oxidative stress induced by PCBs have been studied extensively, and the effects are widely accepted. Likewise, PCB-induced morphologic and functional changes to mitochondria are well documented [35
]; the depolarization of mitochondrial membrane potential and inhibition of ETC activity following PCB exposure have been shown in vitro and in vivo [38
]. The discovery of nonlegacy (non-Aroclor) PCB congeners, such as PCB11, in current commercial goods (e.g., paint, inks, textiles) has once again focused the attention of the scientific community on PCBs [3
]. In 2013, we reported the existence of hydroxylated PCB11 metabolites, including 4OH-PCB11, in circulating human blood [8
]. Because of the relatively short history of research on nonlegacy PCBs, our knowledge of their biological effects on mammalian metabolism is limited.
The mitochondrion is a major target for PCBs and PCB metabolites. We showed that PCBs and their metabolites induced oxidative stress by increasing mitochondrial superoxide (O2
) and hydrogen peroxide (H2
]. Chronic exposure to PCBs and their metabolites significantly altered antioxidant enzyme profiles, including the activity of manganese superoxide dismutase (MnSOD), with no changes in protein level [8
]. Notably, SIRT3, the primary mitochondrial NAD+-dependent deacetylase, regulates the enzymatic activity of MnSOD post-translationally in response to exogenous stress [13
]. Collectively, these results strongly suggest that SIRT3 could play a significant role as an “adaptive-response protein” during PCB exposure because it can regulate mitochondrial function and oxidant detoxification processes by removing acetyl groups from lysine-modified proteins (e.g., increased MnSOD activity via deacetylation) responsible for maintaining redox homeostasis.
In the current study, we observed that the cellular effects of 4OH-PCB11 were more pronounced in SIRT3-KO MEFs than in WT. Short-term (24 h) treatment with 3 μM 4OH-PCB11 significantly increased the doubling time of SIRT3-KO cells (by decreasing cell growth), while WT cells were not affected. Similarly, this metabolite also decreased the mitochondrial membrane potential of SIRT3-KO MEFs, suggesting that 4OH-PCB11 may influence mitochondrial function and lead to alterations in oxidative metabolism. Interestingly, no cells displayed an increase in the steady-state levels of mitochondrial O2
following 4OH-PCB11 treatment, although we did not assess the flux of superoxide radical formation and the data only represented a single time point (24 h) immediately following 4OH-PCB11 treatment. To determine whether MnSOD, the major O2
detoxifying enzyme in mitochondria, responded to 4OH-PCB11, we assessed its abundance and enzymatic activity. Under basal conditions, MnSOD activity was lower in SIRT3-KO MEFs than in WT MEFs, while exhibiting similar levels of MnSOD protein, see Figure 3
A. Treating WT MEFs with 3 μM 4OH-PCB11 increased MnSOD enzyme activity, but this was not accompanied by an increase in MnSOD immunoreactive protein, suggesting that PCB increased O2
levels to trigger a stress response by mitochondrial SIRT3, see Figure 3
A,B. We reason that SIRT3 could deacetylate the critical lysines in MnSOD to increase its enzymatic activity, facilitating the removal of 4OH-PCB11-induced O2
while maintaining a steady-state level of MnSOD protein.
To further investigate the effects of 4OH-PCB11 on mitochondria, we assessed cellular respiration with the XF96 Extracellular Flux Analyzer. Because 4OH-PCB11 decreased the growth rate of SIRT3-KO MEFs, we also expected the OCR to decrease. On the contrary, the basal and ATP-linked OCRs increased in PCB-treated SIRT3-KO cells. Moreover, the reserve respiratory capacity (i.e., the difference between maximal and basal OCR) decreased drastically in SIRT3-KO cells treated with 4OH-PCB11. This observation strongly suggests that 4OH-PCB11 treatment in the absence of SIRT3 produces cellular stress, perhaps by damaging mitochondrial DNA or electron transport chain (ETC) complexes, requiring additional energy to respond to inefficiency in oxidative phosphorylation. To provide for this excess energy demand, it is likely that SIRT3-KO MEFs increase their OCR by exhausting their reserve respiratory capacity. SIRT3 targets numerous mitochondrial ETC complexes [45
] to regulate the activity of the complexes and the rates of electron flow. Thus, the damaging effects of stressors such as 4OH-PCB11 could be exacerbated in the absence of functional SIRT3.
Another mechanism that mammalian cells could use to respond to exogenous stressors like PCBs is metabolic reprogramming. For example, when energy metabolism is perturbed by genotoxic stress, fatty acid oxidation (FAO) can be initiated as an adaptive response [46
]. Because SIRT3-KO cells demonstrated decreased growth and increased OCRs (at the expense of their reserve respiratory capacity), we explored whether 4OH-PCB11 treatment also altered FAO-driven respiration or the expression of genes involved in regulating fatty acid metabolism. We found that basal levels of endogenous FAO-associated oxygen consumption decreased significantly in SIRT3-KO MEFs compared to WT, which is consistent with information in the literature [32
] demonstrating that SIRT3 targets FAO enzymes for deacetylation. Interestingly, when SIRT3-KO and WT cells were treated with 3 μM 4OH-PCB11 for 24 h, the FAO-associated OCR decreased in WT MEFs; however, SIRT3-KO MEFs demonstrated an opposite response to 4OH-PCB11 treatment by doubling their endogenous FAO-driven OCR. This suggests that there are additional mechanisms by which PCBs can alter substrate utilization in the absence of SIRT3. We also cannot disregard the possibility that mitochondria become uncoupled in these cells with limited substrate media. FAO-associated OCR measurements performed with exogenous substrates (BSA-conjugated palmitate) revealed similar results for vehicle-treated WT and SIRT3-KO MEFs, where KO cells had a significantly lower OCR than WT cells. However, when both WT and SIRT3-KO MEFs received 3 μM 4OH-PCB11 for 24 h, the exogenous FAO-associated respiration decreased (more drastically in SIRT3-KO cells), implying that 4OH-PCB11 could affect long-chain fatty-acid transport into the mitochondria. It should also be noted that some of our results contradicted previously published data [49
]. The differences in culturing conditions in each study, specifically oxygen levels, must be taken into consideration while interpreting the results in these studies.
Because our FAO respiration studies (especially exogenous FAO results) were not conclusive about the effects of 4OH-PCB11 on metabolic reprogramming in the presence and absence of SIRT3 function, we next assessed the expression of genes involved in regulating fatty acid metabolism. Treatment with 3 μM 4OH-PCB11 for 24 h significantly increased the expression of the following genes in SIRT3-KO cells: Acsbg2, Acsm2, Acsl1, Acot12, Hmgs2, Oxct2a, Gk2, Lpl, Slc27a5, and Fabp1. Fatty acids play many important roles, especially as an energy source and as building blocks for triacylglycerols, cholesteryl esters, phospholipids, and the signaling molecule diacylglycerol. These processes start with a common “activation” step in which fatty acids are converted to fatty acyl CoA by acyl-coenzyme A synthetases [50
]. When treated with 4OH-PCB11, SIRT3-KO cells displayed significant increases in the expression of three acyl-CoA synthesases: Acsbg2, Acsm2, and Acsl1. Long-chain fatty acid-CoA ligase (Acsbg2) facilitates the activation of long-chain fatty acids for both the synthesis and degradation of cellular lipids. Mitochondrial acyl-CoA synthetase (Acsm2) and long-chain fatty-acid CA ligase 1 (Acsl1) activate long-chain and medium-chain fatty acids, respectively. We also observed an increase in the expression of acyl-coenzyme A thioesterase 12 (Acot12), which is responsible for hydrolyzing acetyl CoA to acetate and CoA; it plays a key role in the synthesis of cytosolic acetyl-CoA, which in turn is important for the synthesis of fatty acids and cholesterol [51
Two genes involved in ketone metabolism, mitochondrial succinyl-CoA:3-ketoacid coenzyme A transferase 2A (Oxct2a) and hydroxymethylglutaryl-CoA (HMG-CoA) synthase (Hmgs2), were both upregulated in SIRT3-KO MEFs following treatment with 4OH-PCB11. Oxct2a, involved primarily in spermatogenesis, catabolizes ketone bodies and transfers CoA moieties from succinate to acetoacetate [54
], while Hmgs2 condenses acetyl-CoA and acetoacetyl-CoA during cholesterol synthesis and ketogenesis [55
]. Additionally, 4OH-PCB11 increased the expression of genes related to triacyglocerol metabolism, lipoprotein lipase (Lpl) and glycerol kinase (Gk2), which regulate the hydrolysis of triglycerides making up circulating chylomicrons and the degradation of glycerol to glycerol phosphate. The upregulation of these genes is associated with metabolic diseases such as diabetes [56
]. The last two genes that were upregulated by 4OH-PCB11 were bile acyl-CoA synthetase (Slc27a5) and fatty-acid binding protein (Fabp1), which bind and transport long-chain fatty acids into cells. Slc27a5, a member of solute carrier family 27, can also mediate the entry of secondary bile acids into the liver. On the other hand, Fabp1 can bind to cholesterol, bilirubin, fatty acids, and their coenzyme A derivatives [58
]. In light of several reports demonstrating elevated serum triglycerides and cholesterol levels in PCB exposed populations [60
] our results could propose a molecular link between PCB induced mitochondrial dysfunction and their metabolic consequences in humans, including increased incidences of diabetes, cardiovascular disease, and hypertension.
Finally, we investigated whether there was a causative relationship between the SIRT3-mediated increase in MnSOD activity in response to 4OH-PCB11 and PCB-induced effects on FAO-related gene expression and mitochondrial function. When we overexpressed MnSOD in SIRT3-KO MEFs with and without 4OH-PCB11, we found no significant differences in the expression of the following six genes: Acot12, Acsl1, Gk2, Hmgs2, Lpl, and Oxct2a, suggesting increased MnSOD activity was able to revert the increased gene expression of genes associated with fatty acid biosynthesis, metabolism, and transport. Overall, our results demonstrate that MnSOD activity plays a significant role in regulating mitochondrial function as well as the expression of genes involved in regulating fatty acid biosynthesis, metabolism, and transport during exposure to 4OH-PCB11.