Toxic Mechanism Of N-nitroso Compounds On Esophageal Cells And The Intervention Effect Of Vitamin C

Objective:（1）To investigate the exposure level of N-nitroso compound（NOC）precursor nitrite and plasma vitamin C（VC）levels in villagers with high incidence of esophageal cancer in Huaian,Jiangsu.（2）Human normal esophageal epithelial cells（HEEC）was exposed to N-methyl-N’-nitro-N-nitrosoguanidine（MNNG）,to investigate its toxic effect on esophageal cells and to further explore the related mechanisms.（3）HEEC was interfered with different concentrations of vitamin C and MNNG at the same time,to explore the effect of vitamin C on MNNG-induced esophageal cytotoxicity.Methods:（1）Questionnaire was used to collect the general situation,life habits and dietary frequency of the surveyed subjects,three days of diets were collected by double meal method to detect the content of dietary nitrite,and blood samples were collected to detect the content of plasma nitrite and vitamin C.The indexes were compared with the existing standards and their relationship with social demographic characteristics and part of dietary frequency were analyzed.（2）After HEEC was exposed to MNNG at 0,5,10,20,40,80,and 160μmol/L for 24,48,and 72 h,the effect of MNNG on the cell activity of HEEC was detected,then HEEC was exposed to MNNG at 0,5,10,and 20μmol/L for 24 h,the effects of MNNG on membrane integrity,total intracellular ROS levels were detected.Apoptosis and cell cycle were detected by flow cytometry.Real-time fluorescence quantitative PCR was used to detect the m RNA expression of P53,Bcl-2/Bax,CDC25C,Cyclin B₁,CDK1,and CDC25A genes related to apoptosis and cell cycle.The expression levels of P53,CDC25C,Cyclin B₁,CDK1 and p-CDK1（Tyr 15）proteins were detected by Western blot analysis to explore the related mechanism.（3）The CCK8 method was used to detect the effects of 0,1,10,100,and1000μmol/L vitamin C intervention,as well as the effects of different concentrations of vitamin C and 20μmol/L MNNG intervention on the proliferation of HEEC cells,followed by 24 hours of intervention with 0,0.1,1,10μM vitamin C and 20μmol/L MNNG,intracellular ROS,apoptosis and cell cycle were detected to observe the inhibitory effect of vitamin C on HEEC toxicity induced by MNNG.The m RNA expression of P53,Bcl-2/Bax,CDC25C,Cyclin B₁ and CDK1 was detected by real-time fluorescence quantitative PCR.The expression of P53,CDC25C,Cyclin B₁,CDK1 and p-CDK1（Tyr 15）protein was detected by Western blot analysis to explore the mechanism of vitamin C action.Results:1.Nitrite exposure and vitamin C intake levels in high-risk areas of esophageal cancer in Huaian,Jiangsu:there were 184 people in this survey,of whom 51.1%were males.There were significant differences in living habits between men and women（P<0.05）,but there was no significant difference in dietary habits and dietary frequency（P>0.05）.There was no significant difference in internal and external nitrite exposure between males and females（P>0.05）.There was no significant difference in internal and external exposure levels of nitrite among different social demographic characteristics（P>0.05）.The over-standard rate of dietary nitrite was 69.6%,and the difference was not statistically significant between men and women（P>0.05）.The intake frequency of pickled products and the source of drinking water had no effect on the over-standard rate of dietary nitrite and the content of plasma nitrite（P>0.05）.Plasma vitamin C deficiency accounted for 42.4%,deficiency accounted for23.9%,and normal accounted for 33.7%.And it was found that the difference of plasma vitamin C intake with different intake frequency was statistically significant（P<0.01）.2.Study on the toxicity and mechanism of MNNG to HEEC:（1）After HEEC was exposed to different concentrations of MNNG for 24,48,72 h,the cell viability was decreased gradually with the increase of dose,in time-dependent and dose-dependent manner.（2）After 24 h exposure to 0,5,10 and 20μmol/L MNNG,the release of LDH was increased significantly with the increase of concentration,and compared with the control,the difference was significant.（3）ROS detection showed that MNNG significantly increased the production of ROS,and the fluorescence intensity of ROS in 10μM and 20μM MNNG groups was significantly different from that in the control group.（P<0.05）.（4）The results of apoptosis detection showed that MNNG promoted the apoptosis of HEEC,and the total apoptosis rate of each group was significantly increased（P<0.05）,which in a dose-dependent manner.（5）The results of cell cycle detection showed that compared with the control group,the number of cells in G0/G1 phase was decreased and the proportion of S phase was increased in all exposed groups,and the difference was statistically significant（P<0.05）,while the number of S phase cells and G2/M cells in 20μmol/L MNNG group were significantly higher than those in control group（P<0.05）.（6）RT-q PCR detection showed that the m RNA expression of P53 was increased,the expression of Bcl-2/Bax m RNA was decreased,the difference were significant between10,20μmol/L MNNG groups and the control group（P<0.05）.Compared with the control group,the expression of CDC25C m RNA was decreased（P<0.01）in each group,the expression of Cyclin B₁m RNA was decreased in each group（P<0.05）,the expression of CDK1 m RNA was increased in each group（P<0.01）,and the expression of CDC25A m RNA was increased（P<0.05）in each group.（7）The results of Western blot analysis showed that the expression of P53 protein was increased gradually with increasing concentration,the protein expression of CDC25C was decreased,the protein expression of Cyclin B₁ was decreased gradually,10,20μmol/L MNNG groups was significantly higher than that of the control group（P<0.05）.While the protein expression of CDK1and p-CDK1 was increased,and the protein expression of each group was significantly higher in the control group（P<0.05）.3.The effect of vitamin C on HEEC cells exposed to MNNG and its mechanism:（1）After HEEC was exposed to different concentrations of vitamin C,it was found that vitamin C had no significant effect on the activity of HEEC（P>0.05）.Different concentrations of Vitamin C and 20μmol/L MNNG were treated at the same time,and it was found that vitamin C can inhibit the effect of MNNG on the activity of HEEC,and the difference was statistically significant when the concentration was1,10μmol/L（P<0.05）.（2）HEEC was treated with 0,0.1,1,10μmol/L vitamin C and 20μmol/L MNNG at the same time.The detection of intracellular total ROS showed that vitamin C intervention could reduce the ROS produced by MNNG,fluorescence intensity was decreased（P<0.05）.（3）Vitamin C can also reduce the total apoptosis rate which increased by MNNG（P<0.05）.（4）The results of cell cycle detection showed that compared with MNNG exposure group,the 1μmol/L VC+MNNG group and the 10μmol/L VC+MNNG group had more G0/G1 phase cells（P<0.01）,the proportion of S phase in each group was reduced（P<0.05）,and G2/M cells were also reduced（P<0.05）.（5）The results of RT-q PCR showed that vitamin C intervention can reduce the m RNA expression of P53.The 1μmol/L VC+MNNG group and the 10μmol/L VC+MNNG group were significantly lower than the MNNG treatment group（P<0.01）,even lower than the control group（P<0.05）.Compared with the MNNG exposure group,Bcl-2/Bax m RNA was increased in 1μmol/L VC+MNNG group,10μmol/L VC+MNNG group（P<0.05）.The m RNA expression of CDC25C gene was increased after vitamin C intervention,and the three groups containing vitamin C were significantly higher than those of MNNG treatment group alone（P<0.01）.The expression of Cyclin B₁ m RNA in the vitamin C intervention group was higher than that in the MNNG treatment group alone,and the difference was statistically significant（P<0.01）.Compared with MNNG exposed group,the expression of CDK1 of 0.1μmol/L VC+MNNG group significantly was increased,the expression of CDK1 of 1μmol/L VC+MNNG group,10μmol/L VC+MNNG group was decreased,the difference was statistically significant（P<0.01）.（6）Western blot analysis results showed vitamin C reduced the expression of P53 protein up-regulated by MNNG（P<0.01）.Compared with the MNNG exposure group,the CDC25C protein expression in the vitamin C intervention group was slightly increased,but the difference was not statistical significance（P>0.05）.After being exposed to MNNG,the expression of Cyclin B₁ protein was significantly reduced（P<0.01）.In the group with vitamin C intervention,the expression of Cyclin B₁ protein was gradually increased.The 10μmol/L VC+MNNG group was significantly higher than the MNNG exposure group（P<0.01）.Compared with the blank control group,the expression of CDK1 protein in the MNNG exposed group was increased slightly,and the difference was not statistically significant（P>0.05）,but the expression of CDK1 protein in the 0.1μmol/L VC+MNNG group increased significantly（P<0.01）,while CDK1in the 1μmol/L VC+MNNG and 10μmol/L VC+MNNG groups was decreased gradually,and the protein expression in the 10μmol/L VC+MNNG group was decreased to lower than that in the MNNG exposure group（P<0.01）.The expression of p-CDK1（Tyr 15）protein showed a similar trend to that of CDK1,it increased first and then decreased,it was also the highest in the 0.1μmol/L VC+MNNG group.The difference was that the 1μmol/L VC+MNNG and 10μmol/L VC+MNNG groups lower than the expression level in the control group（P<0.01）.Conclusion:（1）The exposure of nitrite in Huai’an area of high incidence area of esophageal cancer is excessive,and the deficiency of vitamin C is serious.The content of vitamin C in plasma is related to the intake frequency of pickled products.（2）MNNG inhibits the vitality of HEEC in a time-and dose-dependent manner.It can also cause the destruction of cell membrane integrity,increase the production of intracellular ROS,and promote apoptosis,which is dose-dependent.The effect of promoting apoptosis is related to the up-regulation of P53 expression and the down-regulation of Bcl-2/Bax m RNA expression.Cell cycle was arrest in G2/M phase by MNNG through the CDC25C/CDK1/Cyclin B₁ pathway,which is reflected in the downregulation of Cyclin B₁,CDC25C protein expression,and the upregulation of CDK1 and p-CDK1（Tyr15）protein expression.（3）Vitamin C had no effect on the viability of HEEC,Vitamin C could reduce the production of ROS,reduce the inhibition of MNNG on cell viability,inhibit its apoptosis,and reduce the arrest of G2/M phase.The inhibition of apoptosis is related to down-regulation of P53 expression and up-regulation of Bcl-2/Bax m RNA expression.increase G0/G1 phase cells.The main way to reduce cycle arrest is to regulate CDC25C/CDK1/Cyclin B₁ pathway,which is embodied in the down-regulation of Cyclin B₁ and p-CDK1（Tyr15）protein expression,the increase of CDK1 and p-CDK1（Tyr15）protein expression induced by MNNG.