| Formaldehyde (FA) is a well-known environmental carcinogen. Since Rapoporty first reported that FA could induce mutation in 1946, quite a lot of researches on the mutation and carcinogenesis of FA have been carried out. Till now, most of the researches focused on exogenous FA, which is an environmental pollution. Recently, it was proposed that endogenous FA may be involved in endothelial cells damage, and may be a potential factor of vulnerability of atherosclerosis. Methylamine (MA) exists in human's blood, urine and tissues. Endogenous FA and an equal molar concentration of hydrogen peroxide (H2O2) are derived from the deamination of MA. In the present study, we used comet assay and K-SDS precipitation assay to detect DNA damage of human umbilical vein endothelial cell line CRL-2480 (HUEC) treated with FA, H2O2, equal molar concentration of FA and H2O2, and investigated whether there exists semicarbazide-sensitive amine oxidase (SSAO) in HUECs with high performance liquid chromatography (HPLC). Main procedures and methods1. Comet assay was employed to assess DNA damage of HUECs treated with either various concentrations (0, 5, 10, 25, 50 ,100μmol/L) of FA, H2O2 or FA and H2O2 for 20 min, or 25μmol/L FA, H2O2, FA and H2O2 for different duration of time (0, 10, 20, 30min). The fluorescence microscope was uesd to observe DNA damage.2. K-SDS precipitation assay was employed to assess DNA-Protein cross-links (DPC) of HUECs treated with either various concentrations (0, 10, 50, 100, 500, 1000, 2000 μmol/L) of FA or FA and H2O2 for 1.5h, or 50μmol/L, 100μmol/L FA, FA and H2O2 for different duration of time (0, 0.5, 1,1.5,2,4h).3. HPLC was employed to detect the SSAO enzyme activity of HUECs. Results:1.1 After treatment with various concentrations (0100μmol/L) of FA for 20min, the tail moments (TMs) ((x|ˉ) ± s) were 0.032±0.018, 0.272±0.229, 0.328±0.278, 0.343 ±0.309, 0.035 ±0.051, 0.037+0.076, respectively. The TMs((x|ˉ)±s) were 0.032 ± 0.018, 4.83 ± 3.22, 7.57 ± 4.99, 16.54 ± 8.23, 24.61 ± 12.33, 32.88 ± 13.88,respectively, after treatment with various concentrations (0100umol/L) of 20min. The TMs(*±s) were 0.032+0.018, 5.74+4.04, 9.02±6.42, 19.16±8.89, 17.18 + 5.78, 23.36 +11.20, respectively, after treatment with various concentrations of FA and H2O2. After treated with various concentrations of FA for 20min, 5, 10, 25 umol/L TMs increased significantly compared with the control (PO.05). After treated with 50umol/L, lOOumol/L FA TM increased nonsignificantly compared with the control (P>0.05). When treated with FA and H2O2 together, the TMs of 5, 10, 25 umol/L groups increased compared with those treated only with H2C>2? while the TMs of 50umol/L, lOOumol/L groups decreased, compared with those treated with H2O2 only.1.2 The TMs (X +s) were 0.032+0.018, 0.372±0.262, 0.394±0.235, 0.584+0.457, respectively, after treatment with 25umol/L FA for different time (0, 10, 20, 30min). The TMs(*±s) were 0.032 ±0.018, 10.86 ±6.79, 19.09 + 9.02, 27.26 ±12.56, respectively, after treatment with 25umol/L H2O2 for different time (0, 10, 20, 30min). The TMs(*±s) were 0.032 ±0.018, 13.08 ±6.48, 20.81 ±10.70, 17.93± 9.75, respectively, after treatment with 25umol/L FA and H2O2 for different time. TM increased significantly compared with the control when treated with 25umol/L FA, H2O2, FA and H2O2 for various duration of time. When the cells were treated with FA and H2O2, TMs increased at 10 and 20 min time points, compared with which were treated with H2O2 alone.2.1 Incubation of HUECs with 02000umol/L FA resulted in concentration-dependent increases in DPC levels. The DNA in the cross-links fraction(X ±s) were 3.97±4.71, 2.73 ±1.07, 4.15 ±2.71, 6.72 ± 2.08, 9.51 ± 1.55, 18.09 ±2.94, 29.35 ± 3.63 respectively, after treatment with 02000umol/L FA. The DNA in the cross-links fraction^±S) were 3.97±4.71, 5.99±2.52, 5.34±3.89, 6.98±6.16, 9.52+5.11, 5.36±2.66, 6.81 ±4.74, respectively, after treatment with 02000umol/L FA and H2O2. There were no significant difference between experimental groups and control (P>0.05).2.2 After treatment with 50umol/L FA for various duration of time, the DNA in thecross-links fraction (*±s) were 4.07+2.77, 3.96±2.49, 6.24±2.12, 4.15+2.71, 9.82+2.23, 6.32+2.45, respectively, and at 2h time point, the DPC level increased significantly compared with control. After treatment with 50umol/L FA and H2O2 for various duration of time, the DNA in the cross-links fraction (■* + s) were 4.07+2.77, 3.34+2.24, 9.18±2.03, 5.34±3.89, 7.65+3.38, 8.33 + 3.61, respectively, and at lh time point the DPC levels increased significantly compared with control. 2.3 After treatment with lOOumol/L FA for various duration of time, the DNA in the cross-links fraction (* + s) were 4.07±2.77, 5.79+1.89, 7.64 + 8.45, 6.72+2.08, 5.86 + 0.98, 5.39+1.64, respectively. After treatment with lOOumol/L FA and H2O2 for various duration of time, the DNA in the cross-links fraction (X ±s) were 4.07 +2.77,4.81+2.28, 6.11+2.29,6.98 + 6.16, 8.48±2.64, 7.14 + 2.43, respectively.3. SSAO enzyme activity was detected in the HUECs.Conclusion:1. In vitro, lower concentrations (<25umol/L) of FA mainly induced HUECs DNA strand break, while higher concentrations (>500umol/L) of FA mainly induced DPC, and both of them were concentration and time-dependent.2. In vitro, H2O2 induced HUECs DNA strand break, which was concentration and time-dependent.3. There exists SSAO in the HUECs. |
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