On Mechanism Of2,4-dichlorophenol-induced Cell Death In HL7702and MEFs Cells

2,4-dichlorophenol (2,4-DCP), one of the most abundant chlorophenols (CPs), has been widely used to produce herbicides, insecticides and pharmaceutical intermediates. Due to its wide applications, hardly biodegradation, easy bioaccumulation,2,4-DCP can enter into organisms via direct exposure or the food chain, resulting in serious risks to animal and/or human health. Hence, Many countries, including China and United States, have included2,4-DCP onto the list of priority pollutants. To maximally reduce the accumulation of2,4-DCP, most of the studies concentrate on the approaches and methods of the degradation by physicochemical and biological methods, while few studies focus on deciphering the toxic effects and the mechanism of action of2,4-DCP. Cell death is one of the most common and serious form of the cytotoxic effects. Based on the reasons mentioned above, in the present study, human liver7702cells (HL7702) and mouse embryonic fibroblasts (MEFs) derived from normal tissue were used as in vitro models to study2,4-DCP-induced cell death and its potential mechanisms in order to supply more adequate toxicity data and provide a reference for toxic mechanisms and toxicity warning of CPs.The results showed that:1.2,4-DCP (0.50,0.75and1.00mM) could inhibit the cell growth, reduce cell viability and induce significant morphological changes (including cellular shrinkage, cell rounding and cytoplasmic vacuolization) in HL7702and MEFs. After exposure of cells to the same concentrations of2,4-DCP for the same time, HL7702cells are more resistant to2,4-DCP than MEFs cells, which might be ascribed to two reasons. One is that HL7702cells were obtained from liver, a detoxification organ, but MEFs were derived from normal mouse embryos, another is that MEFs per se are more sensitive to exogenous poisons than HL7702cells.2.2,4-DCP could induce apoptosis, necrosis and autophagy in HL7702and MEFs. The morphological characterizations of apoptosis, such as nuclear condensation, formation of apoptotic bodies, were observed via Hochest33258staining. The cell apoptosis ratio was quantitatively analyzed by flow cytometry, which showed that apoptosis was induced by2,4-DCP. The collapse of mitochondrial membrane potential (MMP) and the elevation of Bax/Bcl-2ratio suggested that2,4-DCP activated the mitochondrial apoptosis pathway. Furthermore, the increase of caspase-3activity indicated that2,4-DCP-induced apoptosis might be in a caspase-dependent manner. Both apoptosis and necrosis occurred after exposure of HL7702and MEFs to higher concentration of2,4-DCP (1.00mM) for long-time (48h). Additionally, the present study showed that2,4-DCP induced autophagy, as proved by the formation of autophagosome observed with transmission electron microscopy and the transformation from LC3-Ⅰ to LC3-Ⅱ (increase of LC3Ⅱ/LC3Ⅰ ratio) detected by western blotting.3.2,4-DCP induced overproduction of reactive oxygen species (ROS) resulting in oxidative stress. The up-regulations of Heme oxygenase-1(HMOX1) and nuclear factor-E2-related factor2(Nrf2) mRNA expression and increase of Nrf2protein in the nucleus demonstrated that the HMOX1/Nrf2signaling pathway was involved in2,4-DCP-induced oxidant responses. Therefore, persistent oxidative stress may be an important factor of2,4-DCP-induced cell death.4. The results from transcriptome level displayed that2,4-DCP mainly affected endoplasmic reticulum (ER) function-related biological pathways including sterol/cholesterol biosynthetic and metabolic process, response to ER stress, implying that2,4-DCP cytotoxicity is closely related to ER dysfunction. To explore2,4-DCP induced-ER stress, induction of Bip and CHOP, markers of ER stress, were determined by qPCR and western blotting. We found that2,4-DCP dramatically increased the expressions of Bip and CHOP both at mRNA and protein levels. Since induction of Bip and CHOP represented the activation of the ER stress, we further examined which of the three branches in ER stress were activated by2,4-DCP. The results showed that the up-regulations of IRE1α and ATF6both at mRNA and protein levels were observed, moreover, the activation of IRE1α was monitored with spliced Xbpl mRNA by RT-PCR. Xpblu was significantly cut to Xbpls mRNA in a dose-dependent manner. The levels of eIF2α phosphorylation were significantly increased in cells exposed to2,4-DCP (1.00mM) as early as1h, subsequently, it was lowered and recovered to the normal level. These results mentioned above strongly revealed that2,4-DCP induced all three branches (IRE1α, ATF6and eIF2a) of ER stress.5. Pretreatment of MEFs with sodium tauroursodeoxycholate (TUDCA,1.0mM), an ER stress inhibitor, relieved2,4-DCP-induced apoptosis. Moreover, combination of Sal003(Sal, an inhibitor of dephosphorylation of eIF2a,15.0μM) and2,4-DCP significantly (0.75mM) increased eIF2a phosphorylation levels and dramatically impaired2,4-DCP-induced apoptosis compared with2,4-DCP treated alone.2,4-DCP induces ER stress-mediated apoptosis via eIF2α dephosphorylation. Hence, ER stress is another important reason for2,4-DCP-induced cell death.6. We investigated the effect of2,4-DCP (0.50mM) on global DNA methylation with methylated DNA immunoprecipitation sequencing (MeDIP-seq) in HL7702cells. The results showed that2,4-DCP significantly changed2322genes methylation levels (711genes up-regulation,1611genes down-regulation), compared with control group, after exposure of HL7702cells for24h, ultimately disturbing methylation balance. Among these differential genes, Bip and DAPK1, which are ER death pathway related-genes, displayed different methylation levels. We further assessed methylation levels in promoter region of Bip and DAPK1genes CpG island using bisulfite sequencing PCR. The results showed that the up-regulations of Bip and DAPK1mRNA expression are not associated with total methylation levels of promoter region but site specific hypomethylation levels in2,4-DCP treated cells, which suggested that site specific methylation might regulated their gene expressions. What’s more, DNMT1、DNMT3a and DNMT3b mRNA expression were also elevated by2,4-DCP. Collectively,2,4-DCP-induced epigenetic toxicity (abnormal DNA methylation) might be another important reason for2,4-DCP-induced cell death.In summary, the present study showed that2,4-DCP can reduce cell viability and induce morphological changes, including cellular shrinkage and cell rounding and cytoplasmic vacuolization, and evoke oxidative stress, collapse MMP, trigger ER stress, disturb DNA methylation balance, which together contributed to2,4-DCP-induced, apoptosis, necrosis and autophagy, three ways cell death. Additionally,2,4-DCP induces ER stress-mediated apoptosis via eIF2α dephosphorylation.