It was previously reported that oxidative stress is one of the major mechanisms involved in metabolic disorders associated with obesity [31]. BER is the major defender against DNA damage due to cellular metabolism, including that ROS production, deamination, methylation and hydroxylation [32]. As a DNA polymerase plays a role in the polymerization step, Pol β was recognized as an important element in completing the BER pathway and ensuring the faithfulness of BER. In this study, we initially investigated the effect of OA and PA on Pol β and observed a significant decrease in Pol β activity upon OA treatment. Docking assays and mutant studies identified G189 and S180 in Pol β as important sites involved in the interaction of Pol β and OA. OA competed with DCP via binding to these active sites. As the crucial function of Pol β in BER, we demonstrated that OA treatment repressed BER activity and further induced the abundant oxidative stress and dysfunction of mitochondrial, which contributed to the lipotoxicity and DNA damage in hepatocyte cells (Fig. 6).
As a critical role of Pol β in both SP-BER and LP-BER, Pol β deficiency impedes efficiency of BER and sensitizes cells to oxidative or alkylating agents [33]. We further explored the relationship between FFAs and BER. Here, we found that OA could impede the activity of both SP-BER and LP-BER in some context, whereas a high dose of PA showed some inhibitory effects on LP-BER. Our studies provided insight into a potential inhibitory effect of PA on FEN1, another key protein in the BER, which may explain the decreased efficiency of LP-BER.
Recent studies highlight the interplay between fatty acids, oxidative stress, the BER pathway and liver diseases caused by fatty acids [34]. Harini et al. discovered that HFD-feeding Ogg1−/− mice showed increased adiposity, hepatic steatosis, and dysregulation of fatty acid oxidation [35]. Furthermore, Ogg1−/− mice showed an increase in fatty acid oxidation and fatty acid uptake in muscle [36]. Pre-adipocytes from Ogg1−/− mice exhibited higher differentiation and accumulated more lipids compared to WT mice[22]. Another glycosylase, NEIL1, plays a similar role in the obese pattern and glucose intolerance in animals fed by HFD [37, 38]. On the other hand, some studies have examined the expression profile of genes involved in BER in fatty liver. Mice fed by methionine-choline-deficient (MCD) diet demonstrated the up-regulation of mRNA levels of thymine-DNA glycosylase and APE1. In addition, when mice administered with pioglitazone, a drug that can improve hepatic steatosis and necro-inflammation, the expression of OGG1 and oxidative DNA repair activity were increased [39]. However, very few studies explored the direct link between fatty acids and BER proteins. We have provided a novel inhibitor of Pol β that can help further explore the relationship between fatty acids and BER.
Mitochondria serve as the main organelles for energy storage and fatty acid metabolism in mammalian cells [40]. These organelles play a crucial role in cellular energy homeostasis, including functions such as the electron transport chain (ETC), β-oxidation, ATP production, and ROS generation [41]. The main DNA repair pathway in mitochondria is BER, which is responsible for eliminating oxidative damage in mitochondrial DNA, such as 8-oxo-dG adducts, similar to its nuclear counterparts [42]. However, the presence or absence of a robust Pol β-like enzyme in mammalian mitochondria has been ambiguous. Recently, a growing body of research has provided compelling evidence for the existence of nuclear-encoded Pol β in mitochondria, which actively participates in BER activity [6, 7, 43]. Accordingly, our results showed that increased ROS levels resulting from excessive intake of OA and PA led to abnormal mitochondrial function. It was observed in the experiments that OA and PA induced ROS production during steatosis in HepG2 cells, resulting in increased damaged DNA, which aligns with previous studies [44, 45].
It is well known that liver is the main organ involved in lipid metabolism [46]. Hepatic steatosis derives from an increase in fatty acid, which is associated with elevated lipid peroxidation and decreased excretion of very-low-density lipoprotein (VLDL) [44, 45]. Hepatic cells are particularly susceptible to reactive oxygen species (ROS) exposure, and numerous studies have indicated a significant reduction in hepatic antioxidant systems in various chronic liver diseases. Excessive β-oxidation of fatty acids leading to oxidative stress is considered a contributing factor in several liver diseases, including non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma (HCC). Furthermore, during the development of these FFA-related diseases, DNA damage caused by oxidative stress is recognized as one of the major mechanisms [47]. In a mouse model fed a high-fat diet (HFD), excessive intake of fatty acids resulted in a substantial accumulation of lipids in the liver tissue. Then, we examined damage foci of DNA strand break and found that increasing DNA damage had a direct relationship with fatty acid intake. In agreement with liver tissue, the similar results were observed in HepG2 cells treated with OA:PA, suggesting that treatment of FFAs increased accumulation of lipid and induced DNA damage in vitro.
In conclusion, this study revealed a novel mechanism that FFAs impaired BER activity through repression of key BER proteins and induced the dysfunction of mitochondria. It given additional evidence for the cytotoxicity effects of overloading OA and PA on DNA repair and mitochondrial function.