| Di-(2-ethylbexyl) phthalate (DEHP) is widely used as a plasticizer in manufacture plastic products, which not covalently bound to the plastic matrix. As a result, it can leak out of some plastic products and ubiquitously distribute in the environments, including air, water and soil. DEHP is considered as a ubiquitous environmental comtaminant due to its wide spread use and physical-chemical properties.Adverse effects of DEHP have attracted intensive attention because humans were exposed via inhalation, oral intake or dermal contact to DEHP in the environments. Over the past years, studies have reported the harmful effects of DEHP on the liver maternal, embryo and fetal as well as the thyroid and immune system. However, very little data were reported on the liver damage and molecular mechanism of hepatic toxicity of DEHP in humans. DEHP is metabolized into various metabolites, of which mono-(2-ethylhexyl) phthalate (MEHP) is considered the most toxic one. Previous studies suggest that DEHP as a peroxisome proliferators acts ligand for the peroxisome proliferators-activated receptor (PPAR) which could cause the hepatocytoxicity. Recent studies show that the active PPAR pathway was not only the mechanism of DEHP-induced liver toxicity, which possibly related to the following molecular events, such as inflammatory response, oxidative stress and the activation of oncogenes. Multiple cell signaling pathways and key proteins were involved in DEHP-induced hepatocytoxicity, P53 and sestrins gene family act as important roles in the process of DEHP-induced oxidative damage of the hepatocytes. P13K-AKT-mTOR pathway is closely relevant to the disorder of cell proliferation and apoptosis. Based on these results, two hypotheses for the mechanism of DEHP-induced hepatocytoxicity were provided:one implicates oxidation damage hypothese:DEHP facilitatesβoxidation response, causes accumulation of reactive oxygen species (ROS) and imbalance of oxidoreduction state in the cells as well as the oxidative DNA damage. Another hypothese implicates imbalance of cell proliferation and apoptosis: DEHP causes the cell abnormal proliferation and lead to tumor. The mechanism of the DEHP induced hepatocytoxicity is varied in different cells, the lead role of the above hypothese is still unclealy. Based on the previous results, in the present study, three cell lines including human embryonic liver cell (L02), human liver cancer cell (HepG2) and human liver cancer cell (Hep3B, p53-null) were selected. The cytotoxicities and the regulatory mechanisms of p53 and sestrins in DEHP-induced oxidative damage in the cell lines were investigated; the role of P13K-AKT-mTOR signaling pathway in DEHP-induced Hep3B cell proliferation was investigated as well. The study consists of two parts:Part One:The Regulatory Mechanisms of p53 and sestrins in DEHP-induced Oxidation DNA Ddamage in VitroL02 cells, HepG2 cells and Hep3B cells were treated with DEHP (62.5,125,250, 500 and 1000μM) and dimethyl sulfoxide (DMSO as the solvent control, final concentration<0.1%). According to the experimental design, the treated cells were harvested at the indicated time points, the following indicators were measured:(1) The cell viability assays were carried out at the time points of 12,24 and 48h.(2) The activities of CYP1A1 and CYP3A4 enzymes were detected at the time points of 24, 48 and 72h. (3) The levels of reactive oxygen species (ROS), maleic dialdehyde (MDA), glutathione (GSH) and total anti-oxidation capability (TAOC) were assessed at the time points of 2,4,6,8,12 and 24h. (4) The assessment of cellular 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and cell cycle were analyzed at the time points of 12 and 24h. (5) p53, p73 and sestrins at mRNA and protein levels were detected at the 24h time point. The results are as follows:1. Cell viability(1) At the time points of 12,24 and 48h, DEHP reduced the viability in all treated groups of both L02 cells and HepG2 cells (p<0.05 or p<0.01), excepted for the 62.5μM treated group in 24h in L02 cells. (2) DEHP reduced the viability in all treated groups of Hep3B cells at the time points of 12 and 48h(p<0.05 or p<0.01), but increased the viability in all treated groups of Hep3B cells in 24h, the peak value approximately 1.3-fold in the 250μM treated group (p<0.05).2. Activities of Cytochrome P-450 enzymes(1) DEHP increased the activities of CYP3A4 enzyme in 24h in L02 cells, excepted for 62.5μM treated group (p<0.05 or p<0.01), a peak value approximately 1.4-fold in the 125μM treated group (p<0.01). (2) DEHP increased the activities of CYP3A4 enzyme in 48h in all treated groups of HepG2 cells (p<0.05 or p<0.01), a peak value approximately 5.8-fold in 1000μM treated group (p<0.01). (3) DEHP increased the CYP3A4 enzyme activities in 24h in all treated groups of Hep3B cells, excepted for 62.5μM treated group (p<0.05 or p<0.01), a peak value approximately 2.25-fold in 1000μM treated group (p<0.01).3. Oxidative stress indicators(1) DEHP increased ROS levels in the time course (0-24h) in both L02 cells and Hep3B cells (p<0.05 orp<0.01). Increased ROS level were observed in 24h in 62.5, 125 and 1000μM treated groups of L02 cells and in all treated groups of Hep3B cells, the peak values approximately 1.45-and 1.46-fold in 1000μM treated group of L02 cells and 250μM treated group of Hep3B cells (p<0.01 for both). But ROS levels were significantly reduced in 24h in all DEHP-treated groups of HepG2 cells, the level was decreased by 60% in 62.5μM treated group (p<0.01). DEHP caused the fluctuating increases in MDA activities in L02 cells, HepG2 cells and Hep3B cells in the time course (2-24h) (p<0.05 or p<0.01), the peak values approximately 1.4-fold and one-fold were in 6h in 1000μM treated group of L02 cells, and in 12h in both 62.5μM treated group of HepG2 cells and in 24h in 125μM treated group of Hep3B cells, respectively (p<0.01 for all).(3) Reduced GSH activites were detected in the time course (2-24h) in all treated groups of three cell lines (p<0.05 or p<0.01), the activities decreased by 33,28 and 26% in 24h in 1000μM treated groups of three cell lines (p<0.01 for all). (4) During the time course of 2-24h, the fluctuating changes in T-AOC levels were observed in HepG2 cells and Hep3B cells, the peak values approximately 10-fold and 1.6-fold were in 24h in 125μM treated group of HepG2 cells and in 8h in 250μM treated groups of Hep3B cells. But no effects of DEHP were found in L02 cells (p<0.01 for all). (5) DEHP increased 8-OHdG levels in 24h in all treated groups of three cell lines (p<0.05 or p<0.01), the peak values approximately 1.33-,5-and 7.3-fold were in 500μM treated groups of L02 cells and HepG2 cells as well as in 62.5μM treated group of Hep3B cells(p<0.05 orp<0.01).4. Oxidative stress-related genes at mRNA and protein levels DEHP up-regulated p53, p73 and sestrins mRNA levels in 250 and 500μM treated groups of both L02 cells and HepG2 cells, but down-regulated the mRNA level of sestrins in all treated groups of Hep3B cells.(1) DEHP up-regulated mRNA levels of p53 and p73 in 24h in 250 and 500μM treated groups of both L02 cells and HepG2 cells, the mRNA level reached the highest peak in 24h in 500μM treated groups of these three cell lines (2-fold for p53 and 54-fold for p73 in L02 cells as well as 2.1-fold for p53 and 38-fold for p73 in HepG2 cells, p<0.01 for all). (2) DEHP up-regulated sestrins mRNA level in 24h in 250 and 500μM treated groups of both L02 cells and HepG2 cells, and the mRNA level reached the highest peak in 500μM treated groups of both L02 cells and HepG2 cells (1.8-fold for L02 cells and 9.0-fold for HepG2 cells, p<0.01 for all). (3) DEHP down-regulated the mRNA level of sestrins in 24h in all treated groups of Hep3B cells, which reached the minimum value of 20% in 125μM treated group (p<0.01). (4) Changes of protein expression (P53, P73 and Sestrins) were not detected in these three cell lines.5. Cell cycle analysis(1) DEHP increased the percentages of G1/S phases in 24h in 250,500 and 1000μM treated groups, as well as the percentages of G2/M phases in all treated groups of L02 cells, with a peak values approximately 1.2-fold in 1000μM treated group (p<0.01), but DEHP decreased the percentages of S phases in all treated group, the level decreased by 33% in 1000μM treated group (p<0.01). (2) DEHP decreased the percentages of G1/S phases in 24h in 62.5,125 and 1000μM treated groups, as well as the percentages of G2/M phase in all treated groups of Hep3B cells, the values of G1/S and G2/M phases decreased by 21 and 10% in 1000 and 500μM-treated groups, respectively (p<0.05 or p<0.01). However, DEHP increased the percentages of S phases in all treated groups of Hep3B cells, the peak value increased by 27% in 1000μM-treated group (p<0.01). Changes in the cell-cycle phase distribution in all treated groups of HepG2 cells were not detected.Conclusions:The major results in the first part of this study were summarized as follows:There are differences in DEHP-induced hepatic toxicities in these three cell lines.(1) In L02 cells and HepG2 cells, DEHP increased levels of ROS, MDA and 8-OHdG, and up-regulated p53, p73 and sestrins at mRNA level, but only caused G1/S and G2/M cell cycle arrest in L02 cells.(2) In Hep3B cells, DEHP increased levels of ROS, MDA and 8-OHdG, but down-regulated the sestrins at mRNA level, and accelerated G1/S, G2/M and S phases in the cell cycle, indicating that DEHP induced oxidative DNA damage in p53-null Hep3B cells, which did not promote the normal arrest processes of G1 and G2 phases in the cell cycle and then accelerate the mitosis of cells.Part Two:Regulation of PI3K-AKT-mTOR signaling pathway in DEHP-induced cell proliferationBaed on the major results of Part One of this study and the scientific clues about p53 mutation present in the process of the tumor development, as well as serveal p53-independent pathways involved in mechanisms of the DEHP-induced hepatotoxicity, this part focou on the meachanism of PI3K-AKT-mTOR signaling pathway in DEHP-induced Hep3B cell abnormal proliferation. Hep3B cells were treated with DEHP-only group at the concentrations of 62.5,125,250,500 and 1000μM, co-treated group with DEHP plus LY294002 (phosphatidylinositol 3-kinase inhibitor,10,20,30,40 and 50μM), as well as DMSO (the solvent control, final concentration<0.1%). The following indicators were measured in 24h:cell viability, levels of ROS, GSH,8-OHdG, mitochondrial membrane potential, DNA replication rate (BrdU assays) and phases of cell cycle as well as the mRNA and protein levels of the related genes as pi3k, akt,mtor and p70s6k involved in the PI3K-AKT-mTOR signaling pathway. The results were as follows:1. Cell viability DEHP increasd Hep3B cell viability in 24h in all treated groups with DEHP only, but a decrease in cell viability was observed only in co-treated group with DEHP plus 50μM LY294002. Therefore,50μM LY294002 was choosen in the following experiments to co-treat Hep3B cells with DEHP at the indicatated concentrations.2. Oxidative stress indicators (1) Increased ROS levels were observed in 24h in all treated groups with DEHP only (p<0.01, see Part One of this study). The ROS levels were decreased in all co-treated groups with DEHP plus LY294002. The minium value was in 1000μM co-treated group, which decreased by 30%(p<0.05).(2) DEHP decreased GSH level in 24h in all treated groups with DEHP only (p<0.01, see Part One). The GSH level was increased in all co-treated groups with DEHP plus LY294002, except for 500μM co-treated group. The peak value increased by 1.2-fold in 1000μM co-treated group (p<0.05).(3) DEHP increased 8-OHdG levels in all treated groups with DEHP only (p<0.01). The peak value increased by 7.3-fold in 125μM treated group with DEHP only (p<0.01). No change was observed in all co-treated group with DEHP plus LY294002.3. DNA replication rate DEHP increased DNA replication rates in all treated groups with DEHP only (p<0.05). The peak value increased by 4.5-fold in 1000μM treated group with DEHP only (p<0.01). DNA replication rate was decreased in all co-treated groups with DEHP plus LY294002, except for 125μM co-treated group, the value decreased by 60% in 1000μM co-treated group (p<0.05). 4. Mitochondrial membrane potential level DEHP increased mitochondrial membrane potential levels in 62.5,125, and 250μM treated groups with DEHP only (p<0.05). The peak value increased by 1.17-fold in 250μM treated group with DEHP only (p<0.01). Levels of mitochondrial membrane potential were decreased in 62.5,125,250 and 500μM co-treated groups with DEHP plus LY294002, the value decreased by 29% in 250μM co-treated group (p<0.05).5. Analysis of cell cycle DEHP accelerated the percentages of G1/S phase in 62.5,250 and 1000μM treated groups as well as G2/M and S phases in all treated groups with DEHP only (p<0.05 or p<0.01, see Part One). The percentages of cell numbers in G1/S, G2/M and S phases were back to that of the control group in all co-treated groups with DEHP plus LY294002 (p>0.05).6. Levels of the selected genes at mRNA and protein To investigate the mechanism of PI3K-AKT-mTOR signaling pathway in DEHP-induced hepatic toxicity, the seclected genes (pi3k,akt,mtor and p70s6k) were detected at mRNA and protein levels.(1) DEHP up-regulated pi3k at mRNA level in 24h in 250,500, and 1000μM treated groups with DEHP only, the highest peak reached 3.7-fold in 1000μM treated group (p<0.01). The value decreased by 54% in co-treated group with 1000μM DEHP plus LY294002 (p<0.01).(2) DEHP up-regulated akt at mRNA level in 24h in 62.5,125,500 and 1000μM treated groups with DEHP only, the highest peak reached 7.0-fold in 125μM treated group. The value decreased by 14-fold in co-treated group with 125μM DEHP plus LY294002 (p<0.01).(3) DEHP up-regulated mtor at mRNA level in 62.5,125 and 250μM as well as p70s6k mRNA level in all treated groups with DEHP only, the highest peaks of both genes reached 22-and 19-fold (p<0.01) in the same treated group with 62.5μM DEHP only. The value decreased by 4.4-fold for mtor and 9.5-fold for p70s6k in the same co-treated group with 62.5μM DEHP plus LY294002 (p<0.01).(4)DEHP up-regulated protein levels of p-AKT308, p-AKT473, mTOR and P70S6K in all treated groups DEHP only (p<0.05 or p<0.01), the expression levels of the proteins increased by 1.66-,1.46-,2.43- and 1.90-fold in treated group with 250μM DEHP only (p<0.01, respectively). The lowest levels of the selected proteins decreased by 15,40,60 and 40% in co-treated group with 250μM DEHP plus LY294002 (p<0.01, respectively)Conclusion:the major results of the second part of this study are summarized as follows:(1) In Hep3B cells, DEHP induced oxidative DNA damage, and up-regulated the levels of genes (pi3k,akt,mtor and p70s6k) involved in PI3K-AKT-mTOR signaling pathway at mRNA and protein levels. In addition, DEHP increased levels of mitochondrial membrane potential, DNA replication rate and accelerate the cell cycle.(2)The inhibitor LY294002 could decrease the cellular oxidative stress. However, it could not protect the cells from oxidative DNA damage. Furthmore, LY294002 attenuated DEHP-induced up-regulation of the selected genes(pi3k,akt,mtor and p70s6k) involoved in PI3K-AKT-mTOR signaling pathway at mRNA and protein levels, increase of DNA replication rate and mitochondrial membrane potential level, thus inhibited the cell abnormal proliferation.Base on the results described above, we suggest the mechanism of DEHP-induced the abnormal proliferation in Hep3B cell may be:DEHP was metabolized in the cells to several active metabolites via CYP450 enzymes, with the accumulation of ROS and changes in the mitochondrial membrane potential, which could initiate the oxidative DNA damage and the inhibition of the cell cycle arrest owing to p53-deficient in the cells. Furthermore, excessed ROS could activate PI3K-AKT-mTOR signaling pathway and then accelerate the cell abnormal proliferation.The major results of this study suggest that (1) p53, p73 and sestrins could participate the regulatory mechanisms of DEHP-induced oxidative DNA damge. (2) LY294002 could attenuate DEHP-induced oxidative stress in Hep3B cells, but not protect the cells form the oxidative DNA damage. (3) The abnormal proliferation may be due to co-effects of P53-deficiency and activation of PI3K-AKT-mTOR signaling pathway in the of Hep3B cells. Obviously, under the experimental conditions, unbalance between oxidative DNA damage and the repair as well as the disorder between the cell proliferation and apoptosis may contribute to DEHP-induced hepacytotoxicity, p53 may play a key role in the regulations of the processes above. The innovation points of this study:1. p53,p73 and sestrins genes were involved in the regulatory mechanism of DEHP-induced oxdative DNA damage in the cell lines. P53 could protect the cells from DEHP-induced oxidative DNA damage.2. DEHP-induced the abnormal proliferation in Hep3B cells could associate with co-effects of p53-deficiency and activation of PI3K-AKT-mTOR signaling pathway.
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