| Objective To investigate the relevance between the people who drink Moutai liquor and hepatocarcinogenesis by group case-control study, the effects on the mutation of P53 gene in liver tissues of primary hepatocellular carcinoma (HCC) induced by compound factors in rats and the effect on alcohol dehydrogenase(ADH), acetaldehyde dehydrogenase(ALDH) and Cytochrome P450 2E1(CYP2E1) by Maotai liquor in rats. Methods Select the subjects:consecutive eligible patients were recruited as case group with a verified HCC, local residents who were not suffered from tumor were recruited as control group during January 2010 to December 2011 at 9 hospitals in Guizhou. Diagnostic criteria:The diagnosis of HCC was in accord with the latest HCC diagnosis standard of China Cancer Society,except metastatic liver cancer, inflammatory pseudotumor, autoimmune liver disease, genetic liver diseases,etc. Alcohol exposure standards:Alcohol consumption was defined as more than twice a week, and the alcoholic history was more than one year, Moutai liquor drinkers were divided into drinking completely or drinking incompletely. Comparison method between case group and control group:group case-control study. Questionnaire investigation, physical examination and laboratory examination were conducted by the investigators who were qualified medical doctors with licenses at physician levels or above from the above-mentioned hospitals. In order to use the same standards, we trained doctors before the start of the investigation. Record and organize related survey data, Data were analyzed with the SPSS 16.0 software, differences in distributions of categorical covariates between cases and controls were analyzed using chi-square tests. For the group case-control analysis, univariate and multivariate unconditional logistic regression analysis were performed,OR values and 95% confidence intervals(95% CI) were calculated. Animal model reproduction:10 rates were randomly selected from 130 male SD rats after one-week adaptability feed as normal control group (N), the others were equally divided into four groups (30 rats per group) which were Maotai high-dose group (MG), Maotai low-dose group (MD), liquor high-dose group (PG) and liquor low-dose group (PD). The dose of high-dose group is 10ml/kg, while the low-dose group is 5ml/kg, the groups are made intragastric administration one time every morning (5d/w). These group were with the same feed and freely drank water. At the end of 24w, liver cancer short-term molding were made according to document. Aflatoxin B1 (AFB1) was injected through peritoneal cavity,400μg/kg/time, 1time/d,6times/w, and stop to be used after continuously 2w, and then the feed contains 0.015% difluorene (AAF) was continuously supplied for 2w.2 rats were randomly selected for killing after the ends of 8w,16w,24w with making intragastric administration and then taking out liver, partially fresh livers were stored with-80℃ after weighting, others were fixed by 4% formaldehyde solution, made paraffin sections and stained with HE. After short-term molding, remaining rates were killed, and the specimens were processed the same as before. Pathological examination:Haematin-eosin (HE) staining was used to investigate the pathological features in liver,assisted by the department of pathology of Guiyang Medical College. Connective tissue staining:Masson three-color method was used to investigate the collagen fibers deposition in liver tissue,assisted by the department of pathology of Guiyang Medical College. Serology:Chemiluminescence method was used to investigate the content of hyaluronic acid(HA), collagenIV(Col IV). Investigate the mutation in P53 gene Hepatic tissue DNA was extracted according to instruction of DNA kit. Primer design was composed by Beijing Dingguo Biotechnology Co, Ltd. P53 gene direct sequencing was completed by Beijing Dingguo Biotechnology Co., Ltd. Animal model reproduction:75 male Wistar rats were fed the 3 different intakes of Maotai-flavour liquor or 53% alcohol for 7 days(a feeding per day). Blood was collected from the eye socke of the ratslh and 2h after the last of the feeding,then the rats were killed to take out livers, partially fresh livers were stored with -80℃ after weighting.40 male wistar rats were fed the intake (10ml·kg-1) of Maotai liquor or 53% alcohol (10ml·kg-1) for 12 weeks(a feeding per day,6 feeding per week), after the last of the feeding,then the rats were killed to take out livers, partially fresh livers were stored with -80℃ after weighting. Alanine aminotransferase (ALT), aspertate aminotransferase (AST) in serum were assessed by biochemical methods.Alcohol and acetaldehyde in serum were assessed by headspace gas-chromatographic analysis.Superoxide dismutase,glutathione peroxidase(SOD), malondialdehyde(MDA), alcohol dehydrogenase(ADH) and acetaldehyde dehydrogenase(ALDH) in liver tissue homogenate were determined by assay kits. Haematin-eosin (HE) staining was used to investigate the pathological features in liver, assisted by the department of pathology of Guiyang Medical College. Real time PCR was used to determine expression levels of mRNA of alcohol dehydrogenase-1(ADH-1), acetaldehyde dehydrogenase-2(ADH-2) and CYP 2E1 in liver. Immunohistochemistry was used to determine expression levels of proteins of alcohol dehydrogenase-1(ADH-1), acetaldehyde dehydrogenase-2(ADH-2) and CYP 2E1 in liver. Western Plot was used to determine expression levels of genes or proteins of alcohol dehydrogenase-1(ADH-1), acetaldehyde dehydrogenase-2(ADH-2) and CYP 2E1 in liver. Results In part one, There are significant differences between cases and controls in regarding to cigarette smoking 210 (27.6%),non-alcoholic fatty liver disease 336 (44.1%), alcoholic liver disease 245(32.2%), family history of HCC 141 (16.5%), alcohol consumption 300(39.4%), HBV infection 436 (57.2%), pickled food 290 (38.1%), and economic status 5 years ago 420 (55.1%) in cases,and cigarette smoking 116 (14.5%),non-alcoholic fatty liver disease 160(20.1%), alcoholic liver disease101(12.7%), family history of HCC 40 (5.0%), alcohol consumption 180 (22.6%), HBV infection 82 (10.3%), pickled food 225(28.2%), and economic status 5 years ago 647 (81.1%) in controls,with OR of each variable was 3.520,2.464,4.330,2.219,2.451,19.245,6.212,0.174 respectively, P<0.01. The OR of the drinkers with HBV infection was 96.903. In the people drinking moderately, the OR drinking Maotai liquor completely and in drinking incompletely were 0.331 and 0.775 respectively. In the people drinking excessively, the OR drinking Maotai liquor completely and in drinking incompletely were 1.269 and 5.932 respectively. In part two, positive expression of ASPP1, ASPP2 of normal liquor group is significantly lower than that of Maotai liquor group (P<0.05); within alcoholic liver disease and liver cancer with high iASPP, the ASPP1, ASPP2 are low (P<0.05). Mutation detection of P53 gene:successfully increasing 54 cases of 5～8 exons of P53 gene, base insertion is the main methods of mutation, and others are base mutation and deletion. The mutation rate of Maotai liquor group is 42.3%, while the normal liquor group is 78.3%, the difference has statistical significance (P<0.05). In part three, In the Maotai liquor-fed rats,alcohol and acetaldehyde in serum were declined significantly (P<0.01), superoxide dismutase,glutathione peroxidase and acetaldehyde dehydrogenase in liver tissue homogenate were increased significantly (P<0.01), malondialdehyde decreased significantly (P<0.01), acetaldehyde dehydrogenase-2 in liver was ncreased significantly (P<0.01),compared with alcohol-fed rats.On the anther hand, there were no significant differences between Maotai liquor-fed rats and alcohol-fed rats in regarding to both alcohol dehydrogenase in liver tissue homogenate and alcohol dehydrogenase-1 in liver. Besides,the indexes mentioned abve were significant different among the rats with 3 different intakes of Maotai liquor. (2)Hepatic fatty degeneration and inflammatory cells infiltration were observed in both Maotai liquor group and alcohol group,which were infrequent in Maotai liquor group relatively (P<0.05). Compared with the normal group and alcohol group, levels of SOD and GSH-Px in liver tissue homogenate in Maotai liquor group were higher significantly (P<0.01),meanwhile the level of MDA in Maotai liquor group were lower significantly compared with alcohol group (P<0.01) Compared with alcohol group, levels of mRNA and protein of CYP2E1 were lower significantly (P<0.01). Conclusions HBV infection and pickled food were the most common risks for HCC in Guizhou.Alcohol consumption excessively and cigarette smoking may increase the risk too. Alcohol consumption can increase the risk in the people who infected with HBV. The risk of people who drinked Maotai liquor mainly is relatively lower than that of other drinkers. Long term moderate drinking Maotai, the risk of hepatic fibrosis, hepatocirrhosis and hepatocirrhosis with hepatoma are lower than liqueur, it is possible relevant to the mutation rate of P53 induced by Maotai liquor was lower relatively. Maotai liquor can obviously heighten the activity of acetaldehyde dehydrogenase, so as to reduce the accumulation of acetaldehyde and decrease the toxicity of acetaldehyde. The adverse effect on the body will increase,nevertheless, with alcohol consumption increasing. Maotai liquor consumption(10ml·kg-1)for 12 weeks may bring about alcoholic fatty liver in rats,but the degree was lower than that induced by pure alcohol.It perhaps was on account of the inhibition of CYP2E1 by Maotai factors. |
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