The Effects Of Vitamin B12 Deficiency On Human Hepatocytes Proliferation,Apoptosis And Chromosome Instability In Vitro

Vitamin B12(VB12),also known as cobalamin(Cbl),is a class of water-soluble B vitamins that contain a cobalt atom and a corrin ring.Among the various forms of VB12,methylcobalamin(MeCbl)is an active from of VB12 in mammalian cell metabolism,and cyancobalamin(CNCbl)is the main VB12 form of human dietary supplementation.VB12 is an important micronutrients in the metabolism of One-carbon metabolism(OCM).As a coenzyme of methionine synthetase(MS),VB12 is an indispensable key compound for the synthesis of methionine(Met)and the important methyl donor S-adenosyl methionine(SAM)and plays an important role in DNA methylation.VB12 deficiency or metabolic abnormalities can impede the formation of methyl compounds,resulting in genomic instability events such as DNA methylation and gene expression abnormalities,even increasing risk of neurological developmental,cardiovascular,and neurodegenerative disease.The liver is the main organ for VB12 storage and metabolism.To investigate the effects of different forms of VB12 deficiency on human hepatocyte proliferation,apoptosis,and chromosomal instability(CIN),we selected human normal hepatocytes HL-7702 and human hepatoma cell QGY-7703 as subjects.On the basis of the VB12 concentration of RPMI-1640 medium,the optimum VB12 concentration in maintaining human lymphocyte genomic stability,and the VB12 concentration according to the clinical physiological reference,two cell lines were cultured for 9days in intervention medium of RPMI-1640 containing CNCbl or MeCbl 0,37,370,and 3700 pmol/L in our study.The growth of test cell lines was evaluated by typan blue exclusion,and cytokinesis-block micronucleus cytome assay(CBMN-Cyt)was used to evaluate the nuclear division index(NDI),apoptosis and CIN of two test cell lines.The results of the study show that:(1)Compared with the control group(3700 pmol/L),370 pmol/L CNCbl and MeCbl had no significant effects on the proliferation and CIN of HL-7702 cells and QGY-7703 cells.Both 0 and 37 pmol/L of CNCbl and MeCbl were significantlyreduced cell proliferation rate(P<0.05)and NDI(P<0.05),and significantly elevated CIN(P<0.05)and apoptosis(P<0.05)of two test cell lines.(2)When we compared the effects of CNCbl and MeCbl on the NDI,CIN and apoptosis of the two test cell lines,it was found that the NDI rates of HL-7702 cells cultured with both 37 pmol/L and 3700 pmol/L CNCbl were significantly lower than that of the cells cultured with the same concentrations of MeCbl(P<0.05),while CIN and apoptosis were significantly higher than those cells under the same concentration of MeCbl(P<0.05).In QGY-7703 cell,although cell apoptosis and NDI were no significantly differences between CNCbl and MeCbl at 37 pmol/L,the CIN of cells cultured with 37 pmol/L of CNbl was significantly higher than that of cells cultured with the same concentration of MeCbl in QGY-7703 cells(P=0.003).When the CNCbl concentration was 3700 pmol/L,the NDI rate of QGY-7703 cells was significantly lower than that of cells cultured with 3700 pmol/L MeCbl(P=0.031),while the CIN(P=0.015)and cell apoptosis(P=0.035)ware also significantly higher than cells cultured with 3700 pmol/L MeCbl.These results indicated that both forms of vitamin B12 deficiency could induce chromosomal instability in normal human hepatocytes and hepatoma cells and reduce cell proliferation rate,eventually leading to cell apoptosis.Compared with CNCbl,MeCbl could play better roles in maintaining cell proliferation status,chromosomal stability,and apoptosis of cells.