The Experimental Study On The Molecular Mechanism Of Reduced Coenzyme NADH Anti-mutagenicity

Background:As with many other diseases, both genetic and environmental factors are implicated in the etiology of human cancers. It is now recognized, as you shall learn, that sequential mutations or inappropriate expressions of several different classes of cellular genes [oncogenes, tumor suppressor genes, DNA nucleotide mismatch repair genes, and genes that mediate programmed cell death (apoptosis)] are probably involved in the usual multiple-step process that leads to human cancer. the oncogene theory postulates that the oncogenes of transforming retroviruses are derived from normal cellular genes (proto-oncogenes); and that an increased expression (activation) of proto-oncogenes or an inappropriate expression of mutated forms of proto-oncogenes, occurring spontaneously or induced by cancer-causing agents, contributes to neoplastic transformation and the development of cancer, so means of anti-mutagenesis play a very important role in the prevention of tumorigenesis.Coenzyme NADH involves in energy metabolism and transportation of nucleic acid, proteins and carbohydrates. NADH quinone oxidoreductase 1 (NQ01) is aubiquitous cytosolic flavoenzyme that catalyzes two-electron reduction of various quinones, with NADH or NADPH as an electron donor. This NQ01 -mediated reduction mechanism is responsible for the cellular defense against various damaging oxyradicals. On the basis of its biochemical properties, NADH is the most biological potent antioxidant found in the body and has the ability to protect the body from the damages of free radicals. It is reported that NADH can inhibit production of cellular reactive oxygen species , decline potential of mitochondria. Devoid of NADH in Nerve cell results in denaturalization and necrosis, which is associated with Alzheimer 'diseae. NADH can rescue cell damage induced by rotenone and regulate bcl-2, p53 and bax gene expression.lt is learning that NADH may have a direct positive involvement in enhancing the level of energy metabolism, repairing cellular damage, improving cellular stress ability, decreasing cytotoxicity caused by chemotherapy drugs, chemical toxic agents and radiation. So we want to investigate if there have anti-mutagenesis effects of NADH. Objective:To verify the anti-mutagenesis effect of coenzyme NADH in vitro. To Investigate the potential molecular mechanism of reduced coenzyme NADH anti-mutagenesis from aspects such as mutations of oncogenes, transcription of oncogenes and expression of oncogene proteins. In vivo, to study the protection effects of Reduced Coenzyme NADH on DENA-induced hepatocarcinogenesis rats . Methods:1. Detecting the anti-mutagenesis effect of coenzyme NADH by Ames assay and SCGE assay.2. PCR-SSCP analysis and PCR-RFLP analysis of the mutations of H-ras and p53 in L02 cells induced by DENA, and investigating the anti-mutagenesis effect of coenzyme NADH on them. Investigating the effect of coenzyme NADH on c-erbB2gene amplification by sourthern blot analysis. RT-PCR analysis of p53, rasP21, c-myc,c-erbB2 and ODC mRNA (transcript). Positive rate of p53, rasP21, c-myc, c-erbB2,CyclinDl and ODC proteins expression was determined by flow cytometry. Theenzyme activity of p38MAPK was determined by Western blot.3. Establishing a rat model carrying hepatocellular carcinoma induced by DEN A.Observing the effect of NADH on the hepatocarcinogenesis rate, inhibition rate ofliver cancer, liver coefficient and the survival curve of rats induced by DENA.Detecting the concentration of AFP, y-GT in serum of rats. Observing thehistological structure and the ultramicrostructure of rats in each group. Evaluating theexpression of p53, rasP21 protein by immunohistochemical stained. Analysis thetranscript of related genes in the ras-p38MAPK signal conduction pathway of rats ineach group by RT-PCR.Results:1. Reduced coenzyme NADH has anti-mutagenesis effect and can inhibit mutationsinduced by mutagens and carcinogenic agents1.1 Ames assay Revertants in NADH group lessened compared with mutagen positive group and the inhibition rate raised.1.2 SCGE assay: The rate of tailing cells in NADH high-dose and middle-dose group lessened compared with mutagen positive group,47%. The length of trail is 21.5±2.9^m. The difference is significant (P<0.01) .2.The molecular mechanism of Reduced Coenzyme NADH anti-mutagenesis 2.1 PCR-SSCP analysis: The mutation rate of H-ras exonl and p53 exon7 in L02 cells induced by DENA decreased with NADH protection: The mutation rate of H-ras exonl in DL02-III and DL02-B cells decreased to 25% (3/12) and 50% (6/12) , respectively. The mutation rate of p53 exon7 in DL02-III and DL02-B cellsdecreased to 33.3% (4/12) and 50% (6/12, respectively. The difference is significant (P<0.01) compared with DENA mutagenesis group.2.2 PCR-RFLP analysis: NADH decreased the mutation frequency of the 12th codon of H-ras and the 249th codon of p53 in L02 cells induced by DENA.2.3 Sourthern blot analysis showed: NADH down regulated the amplification of c-erbB2 gene in L02 cells induced by DENA.2.4 RT-PCR analysis showed: The transcript mRNAof p53n rasP21> c-myc> c-erbB2 and ODC of L02 celles were up-regulated in DENA mutagenesis group. and those were decreased obviously after treated with NADH.2.5 NADH down-regulated the expression of p53> rasP21 n c-myc and CyclinDl proteins in L02 cells induced by DENAThe positive rate of the expression of p53, rasP21, c-myc, CyclinDl and ODC proteins in NADH protection group were decreased compared with DENA mutagenesis group (P<0.05), while the expression of c-erbB2 protein was no difference in each group.2.6 The expression of Phosphorylated p38MAPK was increased obviously in L02 cells induced by DENA, while the tendcy was weaken in cells of NADH protection group.3. The protection effects of Reduced Coenzyme NADH on DENA-induced hepatocarcinogenesis rats.3.1 NADH decreased the hepatocarcinogenesis rate of rats induced by DENA.The hepatocarcinogenesis rate of rats in NADH protection group was 33.3%(5/15). which was far lower than it in DENA-induced hepatocarcinogenesis group as 71.4%(10/14).(P<0.01)3.2 NADH decreased the weight of rats liver cancer induced by DENA. The inhibition rate of liver cancer was 62.5%3.3 NADH Prolonged the life span of DENA-induced hepatocarcinogenesis rats.3.4 The concentration of AFP, Y -GT in serum of rats in NADH protection group lower than it in DENA-induced hepatocarcinogenesis group.3.5 NADH inhibited the over-expression of oncogenes protein such as ras, mp53 ,c-erbB2 during the DENA-induced hepatocarcinogenesis.3.6 Compared with DENA-induced hepatocarcinogenesis group , the transcript expression of Raf-1 > sos in rats of NADH protection group were decreased obviously(P<0.01); the transcript expression of c-fos ^ Grb-2 -. she were mild-decreased(P<0.05),while The transcript expression of GAP were increased(P<0.01). there was no difference in the transcript expression of Elk-1 and SRF gene between two group(P>0.05).Conclusion:1. Reduced coenzyme NADH has anti-mutagenesis effect and can inhibit mutations induced by mutagens and carcinogenic agents. Protecting DNA from damadge.2. The possible molecular mechanism of Reduced Coenzyme NADH anti-mutagenesis is related to its regulation of the oncogenes such as p53^ rasP2K c-myc and c-erbB2 in aspects of anti mutations of DNA, expression of transcript mRNA and expression of proteins. NADH put effect on cell cycle by the regulation of CyclinDl and ODC proteins expression.3. NADH puts protective effects on the DENA-induced hepatocarcinogenesis of rats. It decreased the hepatocarcinogenesis rate of rats induced by DENA and obtained a better inhibition rate of liver cancer , therefore prolonged the life span of DENA-induced hepatocarcinogenesis rats. Decreased the concentration of AFP, Y -GT in serum of DENA-induced hepatocarcinogenesis rats. NADH inhibited the over-expression ofoncogenes protein such as ras, mp53 ,c-erbB2 during the DENA-induced hepatocarcinogenesis and regulated the expression of transcript mRNA of some genes related to the ras/MAPK signal-conduct pathway.