Effect Of COFs On Mitochondrial And Energy Metabolism Related Mechanism Research In Hepatocyte

Background and ObjectiveCook oil fumes is one of the most common air pollutants of indoor living environment in our country, and risk factors of female lung cancer. Numbers of fat-soluble compounds of COFs can into the body by respiratory tract and metabolize in the liver, which may produce some liver toxic substances and cause liver cells damage. The main toxic effect of COFs is oxidative damage, in vivo and vitro studies have been confirmed that COFs may restrain the activity of SOD, increase the level of MDA and ROS, and even inhibit the activity of ATPase. The liver as the most important detoxification organ of the body, although animal experience indicated that COFs can cause inflammation, congestion, edema, microelement metabolic disorder in it, but the liver toxicity mechanism of COFs is not clear. Moreover, the study of the effect of COFs on mitochondria function injury is rarely reported as mitochondria is the target of cell oxidative damage. In addition to study the COFs induced liver and mitochondrial function injury, and the mechanism of energy metabolism disorder is discussed as well. Mitochondrian is the place where a cell energy synthesis and energy metabolism disorder is another cytotoxicity outcome which related with oxidative damage and ROS accumμlation. The present research which focused on both liver cell oxidative damage and energy metabolic disorder, is great help to clarify the liver toxicological mechanism of COFs, and also to provide a reference for biological monitoring and etiology explore and prevention in COFs related. Methods1. Effect of COFs on hepatocyte toxic and mitochondrial function.Different doses of COFs treat cμltured human hepatocyte for 24 hours, MTT method to test cells survival rate and then curve fitting used to explore the cell medium lethal concentration following COFs’ s treatment. Then 4 COFs doses and DMSO-control group would choose for the follow-up experiment. After hepatocyte expose to COFs for 24 hours, Laser scanning confocal microscope(LSCM) used to test ROS level, and high performance liquid chromatography(HPLC) used to test ATP, ADP and AMP level in hepatocyte. mitochondrial enzyme activity, openness rate of MPTP, mitochondrial membrane potential( ψm) and mitochondrial respiratory chain complexes(MRCC I~V) would tested by microplate reader, spectrophotometer, LSCM and ultraviolet spectrophotometer.2. Effect of COFs on mechanism of energy metabolism disorder in hepatocyte.After hepatocyte expose to COFs for 24 hours, Real time- Polymerase Chain Reaction used to test the expression levels of energy metabolism related genes. Spearman analysis used to test the correlation between each index.Results1. Effect of COFs on hepatocyte toxic and mitochondrial function.1.1 Effect of COFs on hepatocyte toxic① COFs siginificantly decreased the survival rate of HL7702 hepatocytes(F=365.568, P<0.05), and infected relationship between concentration and cell survival rate accords with logarithm model(r = 0.929). The half inhibition concentration of hepatocytes is 318.10 μg/ml, 95% confidence interal 277.31 to 366.87μg/ml. 4 COFs doses which survial rate greater than 70% used to the follow-up experiment, namely the 20,40,80,160μg/ml COFs and DMSO control group are the finally doses.② In the doses of 40 to 160μg/ml, COFs siginificantly increased ROS level in hepatocytes(F=241.95,P<0.01) and a siginificant positive correlation has been found between COFs-treated groups and ROS level(r=0.978, P<0.01).③Compared with DMSO group, the COFs-treated groups indicated a decrease-trend of ATP, ADP, AMP and TAN level with the obvious different(F=60.16,P<0.01; F=281.05,P<0.01; F=86.90,P<0.01; F=130.37,P<0.01); In the concentration range of DMSO to 40μg/ml, ATP/ADP value siginificantly increased with the rise of doses(F=234.21,P<0.01); Compared with DMSO group, 80 μg/ml COFs group show a increased EC value with obvious different, however the EC value in other dose-groups have no obvious change.1.2. Effect of COFs on hepatocyte mitochondrial function.①COFs-treated groups show a siginificantly inhibited in mitochondria enzyme activity(F=587.185,P<0.05). Compared to DMSO group, the rate of mitochondria enzyme activity from 20 to 160μg/ml COFs-treated groups respectively are 47.26%, 90.30%, 91.40%, 95.83%. ② COFs siginificantly increased the openness rate of mitochondrial permeability transition pore(MPTP)(F=195.3,P<0.05). Compared to DMSO control group, the relative openness rate of MPTP from 20 to 160μg/ml COFs respectively are 23.43%, 33.22%, 45.44%, 56.64%. ③In the concentration range of 40~160μg/ml, COFs siginificantly decreased ψm in HL7702 hepatocytes(F=43.796,P<0.05).④ COFs induced siginificant inhibition of MRCCⅠ~Ⅴactivities, and different COFs-treated groups show a dinfferent degree of decrease on the MRCCⅠ~Ⅴactivities(F=91.673,P<0.01;F=434.581,P<0.01;F=11.904,P=0.01;F=111.063, P<0.01;F=79.111,P<0.01).2. Effect of COFs on mechanism of energy metabolism disorder in hepatocyte.After 24 hours exposure, COFs-induced the expression levels of MRCCⅤmt DNA encode subunits ATPase6 and ATPase8 gene rised siginificantly(F=130.297, P<0.05;F=14.451, P<0.05); the expression levels of MRCCⅣsubunit mt DNA encode gene COXⅠ~Ⅲ m RNA increased siginificantly(F=161.518, P<0.05; F=8.282,P<0.05; F=46.648, P<0.05); the expression levels of micro RNA 181 encoded by n DNA siginificantly increased(F=6.31,P<0.05). Conclusion1. COFs decreased the survival rate of HL7702 hepatocytes and the half inhibition concentration of hepatocytes is 318.10μg/ml, 95% confidence interal 277.31 to 366.87μg/ml. COFs-induced ROS increased and adenosine level decline in hepatocytes.2.COFs-induced mitochondrial enzyme activity decline, increased the openness rate of mitochondrial permeability transition pore, decreased ψm and restrain activities of MRCCⅠ~Ⅴ, Which mitochondrial dysfunction correlation to ROS accumμlation and adenosine synthesis blocked.3. COFs-induced coμld incrased the expression level of ATPase6 and ATPase8 m RNA, restrain the activity of MRCCⅤ and interfered ATP synthesis. Namely, COFs regμlate energy metabolism dysfunction by ATPase6/ATPase8- MRCCⅤ-ATP pathway. COFs regμlate energy metabolism dysfunction by incrasing mi RNA181 gene over-expression and inhibiting the activity of MRCCⅣ and decline its subunit genes encoded by mt DNA COXⅠ~Ⅲ over-expression to reduce ATP level, namely by mi RNA181-COXⅠ~Ⅲ- MRCCⅣ-ATP pathway.4. The alter of mt DNA’s expression caused the decline of MRCCI~V activities and ROS accumμlation, however, ROS woμld against the expression of mt DNA and cause oxidative damage and energy metabolism disorder in turn, namely, the alter of genes related energy metabolism caused the decline of mitochondrial respiratory chain complexes activities is one of the reason of COFs-induced metabolic disorder of energy and oxidative damage in HL7702 hepatocyte.