The Mechanisms Of Tris(2-chloroethyl)Phosphate-induced Hepatotoxicity

Tris(2-chloroethyl)phosphate (TCEP) is a common flame retardant plasticizer. TCEP is often detected in the environmental samples from indoor air, drinking water and household dust) as well as biological samples from zebrafish, gull and breast milk, because it is non-covalently bonded in polymer materials. Experimental studies indicated that TCEP induced neurotoxicity, reproductive toxicity and endocrine toxicity in vitro, and liver tumors in mice. However, little information is available on the molecular mechanisms of TCEP-induced hepatotoxicity.Excessive reactive oxygen species (ROS) are generally generated after exposure of the human body to exogenous toxicants, which damages the biological macromolecules such as proteins, DNA, polysaccharides and lipids. Mitochondria are mainly subcellular organelle of endogenous ROS production. Thus, they are more likely to be attacked by ROS, which cause mitochondrial dysfunction and affect cellular growth and differentiation. It is known that multiple signaling pathways involved in the regulation of cellular growth, of which the PI3K/Akt/mTOR signaling pathway plays a crucial role in controlling cell growth. ROS activated PI3K, which then phosphorylated inositol phosphate and subsequently combined with the PH region of Akt, and provoked the phosphorylation of Akt, thereby regulated cell proliferation and cell growth by activated the downstream effectors of Akt such as FOXO and mTOR. Cellular senescence is an essentially irreversible process of cell cycle arrest. Multiple factors including ROS, mitochondrial damage, telomere shortening and DNA damage can cause cellular senescence through the modulations of the p53-p21-Rb, DNA damage response (DDR) and NFκB signaling pathways. The study aimed to investigate TCEP-induced hepatotoxicity and its mitochondrial toxicity relating to the regulation mechanisms of the PI3K/Akt/mTOR and p53-p21-Rb pathways, in order to provide scientific basis for further explaining TCEP-induced hepatotoxicity.Part One:Investigations of TCEP-induced hepatotoxicityObjectives:The aim of the part was to investigate the TCEP-induced hepatotoxicity.Methods:Chang liver cells, human embryonic liver cells (L02) and human liver cancer cells (HepG2) were treated with the corresponding media containing either TCEP (3.12,12.50,50.00and200.00mg/L) or DMSO (final concentration<0.1%, as solvent control) for24and48h, respectively. We measured the cell viability, lactate dehydrogenase (LDH) release rate, assessed cellular oxidative stress status (including levels of cellular ROS, malondialdehyde (MDA), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), as well as the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio) and analyzed cell proliferation rate, cell cycle distribution and apoptosis.Results:①In TCEP-treated Chang liver cells, the cell viability was decreased in all treatment groups at24and48h compared with the control (P<0.05or P<0.01). The cell viability were respectively, reduced up to81.64%(P<0.05) and75.80%in the200.00mg/L TCEP groups of L02cells at24and48h, compared with the control (P<0.01). The cell viability were respectively, reduced up to87.30%(P<0.05) and88.49%(P<0.01) in the200.00mg/L TCEP groups of HepG2cells at24and48h, compared with the control;②In TCEP-treated Chang liver cells, the LDH release rate was increased in all treatment groups at24h (P<0.05) and to0.48only in the200.00mg/L TCEP-treated group at48h compared with the control (P<0.05). In L02cells, the LDH release rate in all TCP-treatment groups was increased at24h (P<0.05or P<0.01) and in the≥50.00mg/L TCEP groups at48h (P<0.05or P<0.01) compared with the control. In HepG2cells, the LDH release rate was increased in all TCEP-treatment groups at24h (P<0.05) and in the≥12.50mg/L TCEP groups at48h compared with the control (P<0.05or P<0.01);③In Chang liver cells, TCEP increased the ROS level only in the200.00mg/L treatment group at24h compared with the control (P<0.01). In L02cells, TCEP significantly increased the ROS level in the200.00mg/L TCEP group at24h (P<0.05) and in the50.00and200.00mg/L TCEP groups at48h (P<0.05) compared with the control. In HepG2cells, the ROS levels were increased in the50.00and200.00mg/L TCEP groups at24h (P<0.05) and only in the200.00mg/L TCEP group at48h (P<0.05) compared with the control;④In Chang liver cells, the ratios of GSH/GSSG were increased in the≥12.50mg/L TCEP groups at24and48h compared with the control (P<0.05or P<0.01). In L02cells, the ratio of GSH/GSSG was increased only in the200.00mg/L TCEP group at24h (P<0.05), but no change in the ratio of GSH/GSSG in all treatment groups at48h was found, compared with the control. In HepG2cells, TCEP increased the ratio of GSH/GSSG in all treatment groups at24and48h (P<0.05or P<0.01) compared with the control;⑤In Chang liver cells, TCEP inhibited the cell proliferation in≥12.50mg/L treatment groups at24h (P<0.05or P<0.01) and in all treatment groups at48h compared with the control (P<0.05or P<0.01). In L02cells, TCEP inhibited the cell proliferation in all treatment groups at24and48h compared with the control (P<0.05or P<0.01). In HepG2cells, TCEP inhibited the cell proliferation only in the200mg/L treatment group at24and48h compared the control (P<0.05);⑥In Chang liver cells, TCEP induced cell cycle arrest at G2/M phase in the3.12and50.00mg/L treatment groups at24h (P<0.05) and in all treatment groups at48h compared with the control (P<0.05or P<0.01). In L02cells, TCEP induced cell cycle arrest at G2/M phase in the3.12and200.00mg/L treatment groups at24h (P<0.05), but no change in cell cycle distribution was found in all treatment groups at48h, compared with the control. In HepG2cells, TCEP induced cell cycle arrest at G2/M phase of the other treatment groups except for the200.00mg/L TCEP group at24h (P<0.05or P<0.01), and the cell cycle arrest at G0/G1phase in the200.00mg/L treatment group at48h, compared with the control (PO.05). No apoptosis was found in all treatment groups of the three cell lines (P>0.05).Conclusions:TCEP induced oxidative stress, and then resulted in cell growth arrest, but did not induce apoptosis in these three cell lines.Part Two:Regulations of TCEP-induced cell growth arrest via the mitochondrial-mediated signaling pathwaysObjectives:The regulatory mechanisms of mitochondria, Sirtuins and the PI3K/Akt/mTOR pathway in TCEP-induced cell growth arrest were investigated. Additionally, the roles of SIRT1in TCEP-induced cell growth arrest were further done in L02and HepG2cells.Methods:L02cells and HepG2cells were treated with the corresponding media containing either TCEP (3.12,12.50,50.00and200.00mg/L) or DMSO (final concentration<0.1%, as solvent control) for24and48h, respectively. We measured mitochondrial function (levels of mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potential (MMP), mitochondrial DNA (mtDNA) copy number, cellular ATP, and intracellular free Ca2+), expression of SIRT1, SIRT2and SIRT3at mRNA and protein levels, expression of PI3K, p-PI3K, Akt, p-Akt, mT0)R and p-mTOR proteins. Thereafter, we further treated the cells with either EX-527(a SIRT1inhibitor) alone, TCEP alone and EX-527plus TCEP at the indicated concentrations. The proliferation rate and expression of proteins involved in the PI3K/Akt/mTOR pathway were measured. Results:①In L02and HepG2cells, TCEP reduced the cell growth rates in all treatment groups at24and48h compared the control (P<0.05or P<0.01);②In L02cells, TCEP increased the T-AOC levels in the≥12.50mg/L treatment groups at24h (P<0.05or P<0.01) and in the≥50.00mg/L treatment groups at48h (P<0.05or P<0.01). In HepG2cells, TCEP increased the T-AOC levels in the≥50.00mg/L TCEP-treated groups at24h (P<0.05) and in the≥3.12mg/L treatment groups at48h (P<0.05or P<0.01), compared with the control;③In L02cells, TCEP increased the mtROS levels in the50.00and200.00mg/L treatment groups only at48h (P<0.05or P<0.01). In HepG2cells, TCEP increased the mtROS levels in the≥12.50mg/L treatment groups at24and48h (P<0.05) compared with the control;④In L02and HepG2cells, TCEP decreased mtDNA number in all treatment groups at24and48h (P<0.05or P<0.01) compared with the control;⑤In L02and HepG2cells, TCEP decreased MMP in all treatment groups at24h (P<0.05or P<0.01), but no change in the MMP was found in all TCEP-treatment groups at48h compared with the control;⑥In L02cells, TCEP reduced intracellular ATP level only in the200.00mg/L treatment groups at24h and48h compared with the control (P<0.05). In HepG2cells, TCEP reduced intracellular ATP level only in the50.00and200.00mg/L treatment groups at24h (P<0.05), but no change was found in the intracellular ATP levels in all treatment groups at48h (P>0.05), compared with the control;⑦In L02cells, TCEP increased intracellular free Ca2+concentrations in the50.00and200.00mg/L treatment groups at24and48h compared with the control (P<0.05or P<0.01). In HepG2cells, TCEP increased intracellular free Ca2+concentrations only in the200.00mg/L treatment groups at24h (P<0.05) and in the50.00and200.00mg/L treatment groups at48h (P<0.01), compared with the control;⑧In L02and HepG2cells, TCEP up-regulated the levels of SIRT1mRNA in the>3.12mg/L treatment groups at24h (P<0.05or P<0.01) and in all TCEP-treated groups at48h, compared with the control (P<0.05or P<0.01);⑨In L02cells, TCEP down-regulated the levels of SIRT2mRNA in all treatment groups at24and48h compared with the control (P<0.05or P<0.01). In HepG2cells, TCEP down-regulated the levels of SIRT2mRNA in the>3.12mg/L treatment groups at24and48h compared with the control (P<0.05or P<0.01);⑩In L02cells, TCEP down-regulated the levels of SIRT3mRNA in all treatment groups at24h (P<0.05or P<0.01), and in the>3.12mg/L treatment groups at48h, compared with the control (P<0.05or P<0.01). In HepG2cells, TCEP down-regulated the levels of SIRT3mRNA in all treatment groups at24h (P<0.05or P<0.01), and in the>3.12mg/L treatment groups at48h (P<0.05or P<0.01);11In L02cells, TCEP up-regulated the protein expression of SIRT1, down-regulated the expression of SIRT3protein in all treatment groups and of SIRT2only in the200mg/L treatment group at24and48h, compared with control. In HepG2cells, TCEP up-regulated the expression of SIRT1protein, down-regulated the expression of SIRT3protein in all treatment groups at24and48h, but no change was found in the expression of SIRT2protein in all treatment group at24and48h, compared with the control;12In L02cells, TCEP up-regulated the expression of SIRT1protein in the≥12.5mg/L treatment groups at24h (P<0.05or P<0.01) and in all treatment groups at48h (P<0.05or P<0.01), compared with the control. In HepG2cells, TCEP up-regulated the expression of SIRT1protein in all treatment groups at24and48h, compared with the control (P<0.05or P<0.01);13In L02cells, TCEP down-regulated the expression of mTOR, p-mTOR, PI3K, p-PI3K, Akt and p-Akt proteins in the≥12.5mg/L treatment groups at24and48h, compared with the control. In HepG2cells, TCEP down-regulated the expression of mTOR, p-mTOR, PI3K, p-PI3K, Akt and p-Akt proteins in all treatment groups at24and48h, compared with the control;14In L02cells, TCEP inhibited cell proliferation in the≥50.00mg/L treatment groups at24h (P<0.05or P<0.01) and in the≥12.50mg/L treatment groups at48h, compared with the control (P<0.05or P<0.01); TCEP with EX-527inhibited cell proliferation in all co-treatment groups at24and48h, compared with the control (P<0.01). In HepG2cells, TCEP inhibited cell proliferation in the>12.50mg/L treated groups at24h (P<0.05or P<0.01) and in the200.00mg/L treatment group at48h, compared with the control (P<0.05); TCEP with EX-527inhibited cell proliferation in the≥12.50mg/L co-treatment groups at24h (P<0.05or P<0.01) and in all co-treatment groups at48h, compared with the control (P<0.05or P<0.01);15In L02cells, TCEP with EX-527down-regulated the expression of mTOR, p-mTOR, PI3K, p-PI3K, Akt and p-Akt proteins in all co-treatment groups at24and48h, compared with the control. In HepG2cells, TCEP with EX-527down-regulated the expression of mTOR, p-mTOR, PI3K, p-PI3K, Akt and p-Akt protiens in all co-treatment groups at24and48h, compared with the control.Conclusions:TCEP induced the mitochondrial dysfunction and resulted in cell growth arrest via the attenuation of SIRT1-independent PI3K/Akt/mTOR signaling pathway.Part Three:Regulations of TCEP-induced senescence-like cell growth arrest via p53-independent p21/Rb pathwayObjectives:The effects of TCEP on cellular senescence and its regulatory mechanisms of the p53-p21-Rb pathway were investigated.Methods:The L02cell line (L02-p53cells) with inhibited p53expression was established by RNA interference. According to the purpose of this part, L02, L02-p53and Hep3B cells were treated with TCEP (3.12,12.50,50.00and200.00mg/L) and DMSO (final concentration:<0.1%, as the solvent control) for24and48h, respectively. We measured the percentages of senescence-associated P-galactosidase (SA-β-Gal)-positive cells, IL-6levels and expression of p53, MDM2, Rb, p-Rb, Ras, p21, IL6R, p38MAPK, p-p38MAPK, NFkB and p-NFkB proteins in the cells. Results:①In L02cells, TCEP increased the percentage of SA-β-Gal-positive cells in all treatment groups at24and48h compared with the control (P<0.05or P<0.01). In L02-p53cells, TCEP increased the percentages of SA-P-Gal-positive cells in the>12.50mg/L treatment groups at24h (P<0.05or P<0.01) and in all treatment groups at48h (P<0.01), compared with the control. In Hep3B cells, TCEP increased the percentages of SA-P-Gal-positive cells in all treatment groups at24and48h, compared with the control (P<0.05or P<0.01);②In L02cells, TCEP decreased IL-6levels in the≥12.50mg/L treatment groups at24h (P<0.05or P<0.01) and in the≥50.00mg/L treatment groups at48h (P<0.05or P<0.01), compared with the control. In L02-p53cells, TCEP decreased IL-6the levels in the≥12.50mg/L treatment groups at24h (P<0.05or P<0.01),and in the>50.00mg/L treatment groups at48h (P<0.05or P<0.01) comapred with the control. In Hep3B cells, TCEP decreased IL-6levels in the>12.50mg/L treatment groups at24and48h compared with the control (P<0.05or P<0.01);③In L02cells, TCEP up-regulated expression of MDM2protein, but down-regulated p53protein in all treatment groups and p38MAPK, p-p38MAPK, NFκB, p-NFκB and IL6R proteins in the≥50.00mg/L treatment groups, up-regulated the protein expression of Rb and p-Rb in all treatment groups, additionally, the up-regulations of p21protein were found in the≥12.50mg/L treatment groups at24and48h. In L02-p53cells, TCEP down-regulated p38MAPK and p-p38MAPK proteins in the≥12.50mg/L treatment groups, down-regulated NFκB, p-NFκB and IL6R proteins in the≥50.00mg/L treatment groups, moreover, the up-regulation Rb, p-Rb and p21proteins in all treatment groups were found at24and48h. In Hep3B cells, TCEP down-regulated p38MAPK, p-p38MAPK and IL6R proteins in the≥12.50mg/L treatment groups, down-regulated NFκB and p-NFκB proteins in all treatment groups, additionally, the up-regulations of Rb, p-Rb and p21proteins were found in all treatment groups at24and48h. Conclusions:TCEP-induced senescence-like cell growth arrest was probably regulated by p53-independent p21/Rb signaling pathway in the cells, but the IL-6/IL6R and p38MAPK/NFκB signaling pathways was probably not required.