The Effect Of Exposing Vascular Endothelial Cells To Low-Dose Cadmium On Small Molecule Metabolic Profiling Of Nitric Oxide Production

ObjectiveTo study the effect of exposing vascular endothelial cells to low level of cadmium (Cd) on the production of nitric oxide and the change of small molecule endogenous metabolites, in order to explore the probable mechanism in nitric oxide production and endothelial homeostasis of low level of Cd exposed HUVEC from metabolites and metabolic pathway.Methods1. Human umbilical vein endothelial cells (HUVEC) were treated with CdCl2 at various concentrations (0,1,5,10 μmol/L).0 μmol/L CdCl2 was selected as a control group; 1,5,10μmol/L CdCl2 were selected as an experiment group. After 12,24,48 h, CCK-8 assay was carried out to determine the cell viability, the morphology of cells was observed by microscope, the level of cadmium in the cell was observed by atomic absorption spectrometry, the level of lactate dehydrogenase, malondialdehyde in the culture medium were observed by colorimetry, the level of superoxide dismutase in the cell was observed by colorimetry, the level of intercellular adhesion molecule-1 in the culture medium was observed by ELISA, the level of nitric oxide in the culture medium was observed by nitrate reductase method.2. HUVEC were treated with CdCl2 at various concentrations (0,10 umol/L).0 μmol/L CdCl2 was selected as a control group; 10μmol/L CdCl2 was selected as an experiment group. After 48 h, small molecule metabolites from experiment group and control group were subjected to analysis by using gas chromatography-time of flight mass spectrometer (GC-TOF-MS), drawn the total ion chromatogram of each group, combination with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthognnal to partial least squares discriminant analysis (OPLS-DA) for the multivariate statistical analysis to look for variability material between the two groups, combination with bioinformatics to find metabolic pathway. The metabolic pathways were in correlation with ultrastructural alterations of HUVEC that were observed with transmission electron microscope.Results1. Compared with the control group,1,5 and 10μmol/L CdCl2 did not significantly affect cell viability, the morphology of cells did not significantly change, Cd concentration in cells was significantly related with administrative dose and treatment time, the level of lactate dehydregenase did not increase. The level of malondialdehyde trended to increase, but the statistical difference was not significant (P>0.05). The level of superoxide dismutase trended to decrease, but the statistical difference was not significant (P>0.05).5 and 10μmol/L CdCl2 exposure induced expression of intercellular adhesion molecule-1 from HUVEC after 48h and the statistical difference was significant (P<0.05). Different dose of Cd inhibit nitric oxide production from HUVEC after 12,24,48h, the statistical difference was not significant only in 1μmol/L CdCl2 (P>0.05).2. Using GC-TOFMS technique, a total of 433 chromatographic peaks were detected,57 of which were variability materials. Compared with the control group, concentrations of L-malic acid, fumaric acid,3-phosphoglycerate, et al, in total 15 variability materials were significantly reduced in experiment group. Concentrations of ornithine, glycine, uracil et al, in total 42 variability materials were significantly increased in experiment group. The variability materials were significantly in correlation with aspartic acid metabolism, citric acid cycle and glycine, serine and threonine metabolism of metabolic pathways (P<0.05). Compared with the control group, the metabolic pathways were in correlation with ultrastructural alterations of mitochondria, nucleus, endoplasmic reticulum, that did not significantly change except a fraction of vacuolized mitochondria in the experimental group.Conclusion1. Low level of Cd inhibited nitric oxide production, and induced expression of proinflammatory response but did not affect oxidative stress.2. The effect of exposing endothelial cells to low level of Cd on the production of nitric oxide homeostasis when organelle ultrastructure did not significantly change. The mechanism was in correlation with Energy metabolism, amino acids metabolism, lipid and fatty acid metabolism disorders.3. Low level of Cd may inhibit tricarboxylic acid cycle and pentose phosphate pathway which lead to reduce NADPH. Inhibiting NADPH production may reduce nitric oxide production from endothelial cells.