Modulations Of P53-dependent And P53-independent Pathways In Mono-(2-ethylhexyl) Phthalat-induced Apoptosis In Cell Lines L02and Hepg2

Di-(2-ethylbexyl) phthalate (DEHP) has been widely used as a plasticizer in manufacture plastic products to increase the material elasticity and flexibility. DEHP is not covalently bound to the plastic matrix. It can leach out of products and contaminate the external environment, including air, water and soil. The three main pathways of exposure to DEHP are ingestion, inhalation and dermal. In recent years, health effects of DEHP in humans have attracted the special attention of the scientific community due to their high production volume and use in a variety of polyvinyl chloride-based consumer products.Accumulated evidence shows that the DEHP administered orally is rapidly metabolized to mono (2-ethylhexyl) phthalate (MEHP). MEHP, a principle metabolite of DEHP, can cause liver toxicity, kidney toxicity, reproductive or developmental toxicity, as well as induce apoptosis in different types of cells in vitro. However, the underlying mechanism remains to be elucidated.MEHP as a peroxisome proliferators acts ligand for the peroxisome proliferators-activated receptor (PPAR) to induce liver toxicity. Recent studies show that the active DEHP metabolite MEHP can activate the three PPAR isotypes. MEHP-induced liver damage not only related to, the active PPARa, but also did the activation of pregnane X receptor (PXR). Apoptosis is an active form of cell death. This process happens in response to a variety of environmental factors or death signals from the extracellular environment, which regulates by multiple apoptosis-related genes. The p53tumor suppressor plays an important role in the regulation of programmed cell death. The active p53transcriptionally modulates its downstream genes, such as bax, bcl-2and puma, which induced apoptosis through the mitochondrial apoptotic pathways. The high expression of p53protein was detected in MEHP-induced germ cell apoptosis. Previous studies showed that the apoptotic process is not only modulated by p53-dependent signaling pathways, but the p53-independent signaling pathways (such as c-Jun N-terminal kinases-mitogen activated protein kinases (JNK-MAPK) pathway and Fas/FasL pathway) may participate in the process. However, it is unknown whether the p53-dependent signaling pathway and/or p53-independent signaling pathway, participates in the regulations of MEHP-induced apoptosis in human liver cell lines.Based on the previous results, two cell lines (human embryonic liver cell (L02) and human liver cancer cell (HepG2)) were used in this study to investigate MEHP-induced oxidative stress and cytotoxicity as well as p53-mediated mitochondrial apoptosis. And the molecular mechanisms of Fas/Fas ligand pathway (one of p53-independent signaling pathways) involving in the apoptosis were further investigated. The study consists of three parts as follows:Part One: MEHP-induced oxidative DNA damage and apoptosis in human liver cellsObjective: To build up the experimental models with MEHP-treted L02and HepG2cells. Methods:Both L02and HepG2cells were treated with serial twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and dimethyl sulfoxide (DMSO as the solvent control, final concentration:<0.1%), respectively. The following indicators were measured:(1) cell viability was detected by MTT assay;(2) levels of maleic dialdehyde (MDA) and activities of superoxide dimutese (SOD) and glutathione peroxidase (GSH-Px) were determined by the corresponding assay kits;(3) levels of cellular8-hydroxy-2'-deoxyguanosine (8-OHdG) were analyzed by high-performance liquid chromatography (HPLC);(4) apoptosis were analyzed by flow cytometry and TUNEL staining. The results are as follows:1. Cell viability(1) At12h after MEHP treatment, the viability of L02cells increased only in50.00μM treatment group (p<0.05); however, at24h, MEHP significantly reduced the viability of L02cells in50.00and100.00μM treatment groups (p<0.05or p<0.01). At36h, greater reductions in the viability of L02cells were found with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01).(2) At12h after MEHP treatment, the viability of HepG2cells decreased in50.00and100.00μM treatment groups (p<0.05or p<0.01). At24and36h, the viability of HepG2cells reduced in a concentration-dependent manner, greater reductions in the viability were found with higher concentrations of MEHP (>25.00μM, p<0.01for all).2. Cellular oxidative damage(1) At12h after MEHP treatment, no changes in MDA contents were found in all treatment groups of cell lines L02cells and HepG2(p>0.05for all). At24and36h after treatment, MEHP significantly increased MDA contents of L02cells with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01). The MDA contents of HepG2cells significantly increased in all treatment groups at24h (p<0.01for all) and the other treatment groups at36h except for6.25μM treatment group (p<0.01for all).(2) At12h after MEHP treatment, no changes in SOD activities were found in all treatment groups in both cell lines (p>0.05for all). MEHP reduced SOD activities of L02cells in all treatment groups at24h and in high concentration groups (≥25.00μM) at36h (p<0.05or p<0.01). At24h after treatment, MEHP reduced SOD activities of HepG2cells only in100.00μM treatment group (p<0.05), and in50.00and100.00μM treatment groups at36h (p<0.05for both).(3) At12and24h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of L02cells (p>0.05for all). At36h, MEHP significantly reduced GSH-Px activities in all treatment groups of L02cells (p<0.05for all), except for6.25μM treatment group; At12h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of HepG2cells (p>0.05for all). At24and36h, MEHP significantly reduced GSH-Px activities of HepG2cells in all treatment groups (p<0.05for all), except for6.25μM treatment group.3. Cellular8-OHdG levels(1) At24and36h after MEHP treatment,8-OHdG levels of L02cells significantly increased in50.00and100.00μM treatment groups at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).(2) At24and36h after MEHP treatment,8-OHdG levels of HepG2cells significantly increased in high concentration groups (>25μM) at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).4. Apoptosis(1) Flow cytometric analysis showed that in L02cells MEHP significantly increased the percentages of the apoptotic cells in50.00and100.00μM treatment groups at24and36h (p<0.01for all). In HepG2cells, MEHP significantly increased the percentages of the apoptotic cells only in100.00μM treatment group at24h (p<0.01), and in high concentration groups (≥25.00μM) at36h (p<0.0for all).(2) The detection of apoptosis with TUNEL showed that apoptotic L02cells (labeled with green fluorescence) markedly increased in50.00and100.00μM treatment groups at24and36h. There was slight increase in apoptotic HepG2cells (labeled with green fluorescence) only in100.00μM treatment group at24h; obvious increase in apoptotic HepG2cells were found in high concentration groups (≥25.00μM) at36h (p<0.01for all). Conclusions:Under the experimental conditions, MEHP induced the oxidative stress and oxidative DNA damage and then induced apoptosis in L02and HepG2cells.Part Two:Regulation of p53-mediated mitochondrial apoptotic signaling pathway in MEHP-induced apoptosis in human liver cellsThe major results from Part One of this study suggested that MEHP induced liver toxicity and apoptosis, which did not depend only on PPAR pathway. Objective:To investigate modulations of PXR-mediated genes and the protein expressions and enzyme activities, as well as p53-mediated mitochondrial pathway in MEHP-induced apoptotic L02and HepG2cells. Methods:Cell lines L02and HepG2were treated with series twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured:(1) Expression of pxr gene mRNA was evaluated by quantitative real-time PCR (qRT-PCR);(2) Expression of CYP3A4protein was detected by western blotting and activity of CYP3A was measured by7-ethoxycoumarin-O-deethylase (ECOD);(3) Expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA and NOXA proteins were detected by western blotting;(4) Levels of cellular ATP were analyzed by HPLC;(5) Contents of Cytochrome c and Smac/DIABLO in cytosol were detected by western blotting;(6) Caspase3,9activities were determined by the corresponding assay kits; The results are as follows:1. Levels of pregnane X receptor mRNA, CYP3A4protein and CYP3A4activity(1) In L02cells, MEHP up-regulated pxr mRNA levels in all treatment groups except for6.25uM treatment group at24h (p<0.05or p<0.01) and in all treatment groups at36h (p<0.05or p<0.01). In HepG2cells, MEHP up-regulated pxr gene mRNA levels in all treatment groups, except for6.25and12.50μM treatment groups at24h (p<0.05or p<0.01) and in all treatment groups, except for6.25μM treatment group at36h (p<0.01for all);(2) In L02cells, MEHP induced expression levels of the CYP3A4protein in50.00and100.00μM treatment groups at24h (p<0.01for both) and in all treatment groups, except for6.25μM treatment group at36h (p<0.05or p<0.01). In HepG2cells, MEHP induced expression levels of the CYP3A4protein in all treatment groups, except for6.25μM treatment group at24h (p<0.05or p<0.01) and in high concentration groups (≥25.00μM) at36h (p<0.01for all).(3) In L02cells, the ECOD enzyme activities were significantly increased in50.00and100.00μM treatment groups at24h (p<0.05or p<0.01), and in high concentration groups (>25.00μM) at36h (p<0.01for all). In HepG2cells, at24and36h after treatment, MEHP induced increases in the ECOD activities in all treatment groups (p<0.05or p<0.01).2. Expression of apoptosis-related proteins(1) In L02cells, at36h after treatment, MEHP decreased levels of MDM2protein in50.00and100.00μM treatment groups (p<0.05for both), but increased levels of p53and phosphorylated p53proteins in all treatment groups, except for6.25μM treatment group (p<0.05or p<0.01). MEHP increased the p53/MDM2protein ratios in all treatment groups, except for6.25μM treatment group (p<0.01for all), and Bax protein levels in high concentration groups (≥25.00μM,p<0.01), but decreased Bcl-2protein levels in50.00and100.00μM treatment groups (p<0.05or p<0.01). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups (p<0.05or p<0.01) as well as Levels of PUMA and NOXA proteins in high concentration groups (≥25.00μM,p<0.05or p<0.01).(2) In HepG2cells, at36h after treatment, MEHP induced the decrease in MDM2protein level only in100.00μM treatment group (p<0.05), but increased p53protein levels in all treatment groups (p<0.05or p<0.01) and phosphorylaed p53protein levels in high concentration groups (>25.00μM,p<0.01for all). MEHP increased the p53/MDM2protein ratios in all treatment groups (p<0.01for all), and Bax protein level only in100.00μM treatment group but decreased Bcl-2protein level (p<0.05for both). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups except for6.25μM treatment group (p<0.05or p<0.01) and levels of PUMA and NOXA proteins in high concentration groups (≥12.50μM,p<0.05or<0.01).3. Indicators of mitochondrial damage indicators MEHP increased levels of cytosolic Cytochrome c and Smac/DIABOL proteins, but decreased the intracellular ATP contents:(1) MEHP decreased intracellular ATP contents of L02cells in high concentration groups (≥25.00μM) at24h (p<0.05or p<0.01) and in50.00and100.00μM treatment groups at36h (p<0.01for all), but decreased intracellular ATP contents of HepG2cells in all treatment groups (p <0.01for all) at24h and in all treatment groups except for6.25μM treatment group at36h (p<0.01for all).(2) In L02cells at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups (p<0.01for all) and Smac/DIABLO proteins in high concentrations (>25.00μM, p<0.05or p<0.01). In HepG2cells, at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment except for6.25μM treatment group (p<0.05or p<0.01) and the Smac/DIABOL protein at all treatment groups (p<0.01).4. Caspase3,9activities(1) At36h after treatment, MEHP significantly increased the activities of Caspase3,9in high concentration groups (≥25.00μM) in L02cells (p<0.05or p<0.01) and in all treatment groups except for6.25μM treatment group in HepG2cells (p<0.05or p<0.01).Conclusions:In cell lines L02and HepG2, MEHP up-regulated the pxr at mRNA level and induced enzyme activity and CYP3A4protein expression, and increases the levels of p53, p-p53, Bax, PUMA and NOXA proteins, but decreased the levels of MDM2and Bcl-2proteins. Additionally, the mitochondrial apoptosis signaling pathway was activated in the MEHP-treated cell lines.Part three:Regulation of Fas/FasL mediated p53-independent apoptotic pathway in MEHP-induced apoptosis in human liver cellsThe major results from Part Two of this study suggested that p53mediated mitochondrial apoptotic pathway in MEHP-treated cell lines L02and HepG2. Objective:In this part, the technique of RNA interference (RNAi) was used to investigate the regulation of p53-independent pathway in HepG2and L02cell lines. Methods:L02, L02-p53, HepG2and HepG2-p53cells were treated with different concentrations of MEHP (50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured at36h: the percentage of apoptotic cells, expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA, NOXA, Fas/FasL and Smac/DIABOL proteins, cytosolic Cytochrome c concentration, the activities of Caspase3,8,9. The results are as follows:1. The p53silence efficiency in L02-p53and HepG2-p53cells by RNA interference(1) To evaluate the p53silence efficiency, qRT-PCR method was used to measure the levels of the decreasing p53in in L02-p53and HepG2-p53cells. The results showed that in L02-p53cells the p53mRNA and protein levels was decreased by83.1%at24h and52.5%at48h, compared with the corresponding negative controls, respectively;(2) in HepG2-p53cells, the p53mRNA and protein levels were decreased by81.4%at24h and52.4%at48h, compared with the corresponding negative controls, respectively.2. The cell Apoptosis(1) At36h after treatment, MEHP increased the percentages of the apoptotic cells of both L02and L02-p53cells in all treatment groups (p<0.01for all). At36h after L02and L02-p53cells were treated with the same concentration of MEHP, the higher percentages of the L02-p53apoptotic cells were found in all treatment groups compared with the L02cells (p<0.01for all).(2) At36h after treatment, MEHP increased the percentages of both HepG2and HepG2-p53cells in all treatment groups (p<0.01for all). At36h after HepG2and HepG2-p53cells were treated with the same concentration of MEHP, the higher percentages of the HepG2-p53apoptotic cells were found in all treatment groups compared with the HepG2cells (p<0.01for all).3. The expression of apoptosis-related proteins(1) At36h after treatment of L02cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2proteins in all treatment groups (p<0.01for all); at36h after the treatment of L02-p53cells, MEHP up-regulated the expression of MDM2, Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but decreased the expression of p53, p-p53and Bcl-2proteins in all treatment groups, compared with the corresponding controls (p<0.01for all). At36h after treatments of L02and L02-p53cells at the same concentrations of MEHP, the results suggested that the expression of MDM2, Fas and FasL proteins were up-regulated in all treatment groups (p<0.01for all), but the expression of p53and p-p53proteins were down-regulated in all treatment groups (p<0.01for all). No changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins were found in all treatment groups of either L02-p53or L02cells (p>0.05for all).(2) At36h after treatment of HepG2cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2protein in all treatment groups (p<0.01for all); at36h after treatment of HepG2-p53cells, MEHP up-regulated the expression of Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but down-regulated the expression of Bcl-2protein compared with the corresponding negative controls (p<0.01for all). No changes in the levels of the p53, p-p53and MDM2proteins were found in all treatment groups of HepG2-p53cells (p>0.05for all). At36h after treatments of HepG2and HepG2-p53cells at the same concentrations of MEHP, the results suggested that the levels of MDM2, Fas and FasL proteins were up-regulated (p<0.01for all), but the levels of p53and p-p53protiens were down-regulated in all treatment groups (p<0.01for all). There was no changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins in all treatment groups of either HepG2"p53or HepG2cells (p>0.05for all).4. The levels of cytosolic Cytochrome c and Smac/DIABOL protein(1) At36h after treatments of L02and L02-p53cells, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups and the Smac/DIABOL protein in100.00μM treatment group compared with the corresponding controls (p<0.01for all).(2) At36h after treatment of HepG2and HepG2"p53cells, MEHP increased the levels of cytosolic Cytochrome c in100.00μM treatment group (p<0.01for all) and the Smac/DIABLO protein in all treatment groups (p<0.05or p<0.01), compared with the corresponding controls.5. The activities of Caspase3,8, and9At36h after treatment, MEHP increased Caspase3,8,9activities in all treatment groups of L02, HepG2, L02-p53and HepG2-p53cells (p<0.01for all). At36h after treatments either between L02and L02-p53cells or between HepG2and HepG2"p53cells at the same concentrations of MEHP, the results suggested that MEHP induced the activities of Caspase3,8, and9in all treatment groups (p<0.05or p<0.01). Conclusions:The modulation of p53-mediated pathway is not only in MEHP-induced apoptosis in cell lines L02and HepG2. Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial apoptotic pathway in MEHP-induced apoptosis of the cell lines L02and HepG2.The innovation points of this study:(1) This is the first report on the modulation of p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.(2) This is the first report on the Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.