Study On The Effect Of Nonylphenol And Its Isomers On NCTC Clone 1469 Cells

4-n-Nonylphenol(4-n-NP), one of the environmental hormones, is widespread in the human daily life. 4-n-NP is difficult to be degraded in the natural environment with strong bioaccumulation capacity and is harmful to overall organs in the body. Numerous efforts have been focused on the studies about the biological effects of 4-n-NP and its isomers on living organism. In the present study, in order to provide theoretical basis for ecological risk assessment of 4-n-NP and its isomers, we assessed the toxicity of 4-n-NP in a liver cell line from mice, NCTC C lone 1469, and investigated the effects of 4-NP isomers on the total bile acid(TBA) secretion of NCTC Clone 1469.Firstly, we investigated the hepatotoxic effect of 4-n-NP on NCTC C lone 1469 cells. Results showed that:(1) cell viability significantly decreased in 60, 80, 100 μmol/L 4-n-NP groups. NCTC Clone 1469 presented nucleus crenulation after 4-n-NP treatment, with being more and more serious over time, and the activities of LDH, ALT and AST in the supernatant were greater than the control group(*p < 0.05 in 20-60μmol/L, 40-60μmol/L and 60μmol/L 4-n-NP groups).(2) 4-n-NP treatment increased the mRNA and protein express ion of ERα and ERβ at 1-40μmol/L.(3) ICI 182,780 did not reduce LDH, ALT and AST activities in the supernatant. These results suggest that toxic effects of 4- n-NP on the liver cells may have no direct relationship with the ER receptor.Secondly, we explored the oxidative stress in NCTC C lone 1469 induced by 4-n-NP based on two aspects of antioxidant enzyme system and peroxide products comprehensively. It was found that:(1) intracellular ROS and MDA levels were increased in 4- n-NP treated NCTC C lone 1469;(2) SOD and C AT activities in 60 μmol/L 4-n-NP groups significantly decreased compared with control group;(3) the release of LDH, ALT or AST from damaged NCTC C lone 1469 was remarkably inhibited by NAC treatment. It is proposed that the cell damage induced by 4-n-NP was due to oxidative stress.Thirdly, we determined whether 4-n-NP induced apoptosis in NCTC C lone 1469 by Annexin V–FITC and PI staining, and western blotting was assessed to investigate the underlying mechanism. We found that:(1) treatment o f liver cells with 4-n-NP caused remarkable activation of caspase-3 and caspase-12;(2) 4-n-NP increased the protein level of Bax and decreased the protein level of Bcl-2 in NCTC C lone 1469;(3) 1-60 μmol/L 4-n-NP induced a dose dependent elevation of cytosolic free Ca2+;(4) the intracellular Ca2+ level decreased remarkab ly in 60 μmol/L 4-n-NP group with Z-DEVD-FMK or Z-ATAD-FMK treatment. These results indicated that 4-n-NP might promote ERS-induced apoptosis in NCTC Clone 1469.At last, Elisa methods and Reverse Transcription-Polymerase C hain Reaction(RT-PCR) were measured to examine the effects of 4-NP isomers on the total bile acid excretion and mRNA expression of CYP7A1, CYP27A1 and CYP8B1 in NCTC Clone 1469. We observed that 60μmol/L 4-NP37、4-NP111、4-NP112、4-NP194 significantly upregulated the total bile acid excretion of live cells and the mRNA expression of CYP7A1, CYP27A1, CYP8B1 and ERα. It could be speculated that the increase of total bile acid excretion in NCTC Clone 1469 induced by 4-NP isomers was related to enhance the mRNA expression of CYP7A1, CYP27A1, CYP8B1 and ERα.All in all, the present study indicated that: 4- n-NP exhibited cell injury on NCTC Clone 1469, including hepatotoxic effect, oxidative stress and cell apoptosis; 4-NP isomers had effects on the total bile acid excretion of NCTC C lone 1469 and its underlying mechanism maybe related to influence the mRNA expression of C YP7A1, CYP27A1, CYP8B1 and ERα.