Protective effect of vitamin C against tetrachlorobenzoquinone-induced 5-hydroxymethylation-dependent apoptosis in HepG2 cells mainly via the mitochondrial apoptosis pathway

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol, and stimulates the accumulation of ROS to trigger apoptosis. The preventive effect of vitamin C (Vc) against TCBQ-induced apoptosis in HepG2 cells is unknown. And there is little known about TCBQ-triggered 5-hydromethylcytosine (5hmC)-dependent apoptosis. Here, we confirmed that Vc alleviated TCBQ-induced apoptosis. Through investigating the underlying mechanism, we found TCBQ downregulated 5hmC levels of genomic DNA in a Tet-dependent manner, with a particularly pronounced decrease in the promoter region, using UHPLC-MS-MS analysis and hydroxymethylated DNA immunoprecipitation sequencing. Notably, TCBQ exposure resulted in alterations of 5hmC abundance to ∼91% of key genes at promoters in the mitochondrial apoptosis pathway, along with changes of mRNA expression in 87% of genes. By contrast, 5hmC abundance of genes only exhibited slight changes in the death receptor/ligand pathway. Interestingly, the pretreatment with Vc, a positive stimulator of 5hmC generation, restored 5hmC in the genomic DNA to near-normal levels. More notably, Vc pretreatment further counter-regulated TCBQ-induced alteration of 5hmC abundance in the promoter with 100% of genes, accompanying the reverse modulation of mRNA expressions in 89% of genes. These data from Vc pretreatment supported the relationship between TCBQ-induced apoptosis and the altered 5hmC abundance. Additionally, Vc also suppressed TCBQ-stimulated generation of ROS, and further increased the stability of mitochondria. Our study illuminates a new mechanism of TCBQ-induced 5hmC-dependent apoptosis, and the dual mechanisms of Vc against TCBQ-stimulated apoptosis via reversely regulating 5hmC levels and scavenging ROS. The work also provided a possible strategy for the detoxification of TCBQ.