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In the Krebs cycle, succinate is oxidized to fumarate by succinate dehydrogenase (SDH), followed by the conversion of fumarate to malate by fumarate hydratase (FH). In cells with defective SDH and FH, the Krebs cycle is congested, respiration impaired and fumarate and succinate accumulates. Several studies have indicated that the accumulation of these substrates are associated with cytotoxicity and oncogenesis. High levels of succinate and fumarate induce hypoxia inducible factor (HIF1A) hydroxylases, leading to the activation of oncogenic HIF pathways. However, the role of HIF as primary inducer of oncogenic change has been questioned, as other non-enzymatic mechanisms have been shown to interfere with cellular metabolism, cell signalling as well as disrupting protein function. Owing to the essential roles that SDH and FH play in cellular energy metabolism, and their associated tumor suppressor capacity, it is vital to understand the biochemical effects resulting from the accumulation of their associated metabolites. Therefore, in this study, we investigated the effect of high concentrations of succinate and fumarate exposure on cell viability, genome integrity and global DNA methylation using a human hepatocellular carcinoma (HepG2) cell culture model. It was found that relatively high concentrations of succinate and fumarate cause a loss of cell viability, which seems to be orchestrated through an apoptotic pathway. Cells exposed to high levels of succinate also presented with elevated caspase 3 and/or caspase 7 levels. In addition, elevated levels of fumarate lead to extensive DNA fragmentation, which may contribute pathophysiologically by inducing chromosomal instability, while succinate demonstrated lower genotoxicity. Furthermore, both succinate and fumarate altered the global DNA methylation patterns via significant DNA hypermethylation. Since numerous studies have reported correlations between aberrant DNA methylation and oncogenesis, hypermethylation may contribute to the oncogenesis observed in cells exposed to high concentrations of these metabolites.

Non-invasive screening that utilizes cell-free DNA (cfDNA) offers remarkable potential as a method for the early detection of genetic disorders and a wide variety of cancers. Unfortunately, one of the most prominent elements delaying the translation of cfDNA analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the origin of cfDNA is complicated by the apparently arbitrary variability of quantitative and qualitative characteristics of cfDNA in the blood of healthy as well as diseased individuals. These factors may contribute to false positive/negative results when applied to clinical pathology. Although many have acknowledged that this is a major problem, few have addressed it. We believe that many of the current difficulties encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. The results obtained in this study indicate that the release of cfDNA from 143B cells is not a consequence of apoptosis, necrosis or a product of DNA replication, but primarily the result of actively released DNA, perhaps in association with a protein complex. Moreover, this study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

Non-invasive screening that utilizes cell-free DNA (cfDNA) offers remarkable potential as a method for the early detection of genetic disorders and a wide variety of cancers. Unfortunately, one of the most prominent elements delaying the translation of cfDNA analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the origin of cfDNA is complicated by the apparently arbitrary variability of quantitative and qualitative characteristics of cfDNA in the blood of healthy as well as diseased individuals. These factors may contribute to false positive/negative results when applied to clinical pathology. Although many have acknowledged that this is a major problem, few have addressed it. We believe that many of the current difficulties encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. The results obtained in this study indicate that the release of cfDNA from 143B cells is not a consequence of apoptosis, necrosis or a product of DNA replication, but primarily the result of actively released DNA, perhaps in association with a protein complex. Moreover, this study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

The most prominent factor that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in the blood of healthy and diseased individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer patients is not only representative of tumor derived DNA, but also of DNA released by healthy cells under different conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant, but likely includes non-mutated chromosomal DNA fragments. This may cause false positive/negative results. Although many have acknowledged that this is a major problem, few have addressed it. We propose that many of the current stumbling blocks encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Results suggest that the occurrence of cfDNA is not a consequence of apoptosis or necrosis, but primarily a result of actively secreted DNA, perhaps in association with a protein complex. This study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.