| Liver cancer is one of the major serious health diseases of the world, every year the number liver cancer worldwide increased of 626 thousand, of which our country accounted for 55%. According to the World Health Organization liver cancer become the third most serious malignancies just behind the digestive system cancer(gastric cancer and esophageal cancer) in china. The multi-stage model of the liver cancer development(hepatitis-liver cirrhosis-liver cancer) has been supported by a wide range of pathological and clinical researches. Cirrhotic patients after a certain period of time can develop into liver cancer.Now that the incidence of liver cancer is a multifactorial, complex multi-step process, including genetic and environmental factors, alcohol and hepatitis B virus. Acetaldehyde has been proved of carcinogenicity in animals, it may be also associated with human tumors. Acetaldehyde effective concentration in the body and duration of time are affected by the frequency of alcohol consumption and alcohol-related metabolic enzymes ADH and ALDH activity.Alcohol dehydrogenases(ADH) and aldehyde dehydrogenase(ALDH) is the most important enzymes involved in alcohol metabolism in vivo. Ethanol metabolized to acetaldehyde by ADH and further metabolized to acetic acid by ALDH. Liver is the most important organ of ethanol metabolism after the stomach and the intestinal absorpt ethanol through the vein into liver. The liver metabolise 80% to 90% of the total ethanol. There has been reported cancer tissue and normal VII tissue showed significant individual differences in the activity of this two kind of enzymes, such as pancreatic cancer, stomach cancer.Our study was designed to investigate total ADH, ADHI, ADHII and total ALDH, ALDH2 activity differences in liver cancer and normal liver specimens and the relationship of metabolism-related gene polymorphisms and enzyme activities. In order to find the possible reasons of liver cirrhosis patients develops to liver cancer. Methods 1 Human liver samplesHuman liver samples with normal liver function were obtained from 74 patients including 26 male samples and 48 female samples. The age ranged from 20 to 75 years(Median:49 years. Smoking history was positive for 8 donors, negative for 8 donors. Human liver samples with liver cancer were obtained from 68 patients including 58 male samples and 10 female samples. The age ranged from 29 to 75 years(Median:52 years)Smoking history was positive for 58 donors, negative for 10 donors. All subjects wrote informed consent and this study was approved by the ethics committee of Zhengzhou University. 2 Genotyping for ADH and ALDH geneGenomic DNA was isolated with DNA extraction kit from human liver tissues. The subjects were genotyped using polymerase chain reaction(PCR) or direct sequence analysis of polymorphisms for ADH1 B rs1229984(G>A), ADH1Crs698(A>G), ADH1 C rs2241894(A>G), ALDH2 rs671(A>G) and ALDH2 rs13306164(C>T). 3 Preparation of human liver specimens supernatantAll specimens were frozen at- 80℃until analysis. The liver tissue(1:8 w /v) was homogenized in potassium phosphate buffer(0.1 mol/l; p H 7.4) for 20 s using an ultrasonicator(Sonoplus HD 70; Bandelin), centrifuged at 14,000 rpm for 20 min, and cooled to 4℃. The supernatant was used to determine ADH and ALDH activities. 4 Determination of total ADH,ADHI,ADHI and total ALDH、ALDH2 activityTotal activity was estimated by the photometric method with p-nitrosodimethylaniline(NDMA), 4-methoxy-1-naphthaldehyde, 6-methoxy-2-naphthaldehyde, aldehyde as substrates. The reaction mixture contained supernatant, substrate, NAD or NADH, and sodium phosphate buffer. In the Determination of the total ALDH activity the mixture also contained 4-methylpyrazole as a specific inhibitor of ADH activity. The fluorescence was read at an excitation wavelength and an emission wavelength on the Spectrofluorimeter. 5 Statistical analysisCalculation of enzyme kinetic parameters and statistical analysis was performed by Graph Pad Prism 5.0 and SPSS 17.0 software, respectively. All data were routinely tested for normality of distribution and equal variance, and for these tests failure, nonparametric methods of statistical analysis were used. A value of P<0.05 was set as statistically significant. Results 1 Normal liver aldehyde dehydrogenases activity and gene polymorphisms 1.1 The total ADH,ADHI,ADHI and total ALDH、ALDH2 activityNormality test results show that the human liver homogenates total ADH, ADHI, ADHII and total ALDH, ALDH2 activity parameters were not normally distributed, so this experiment non-parametric test was used for statistical analysis. The activity of total ADH,ADHI,ADHI and total ALDH 、 ALDH2 were 1.81(0.16~72.12),0.44(0.09~1.26),0.18(0.03~0.5),0.12(0.01~0.31)pmol·min-1·mg-1pr otein;9.21(0.85~18.35) pmol·min-1·mg-1protein. The data displayed a 447.95、14.38 and 15.99、20.97、21.62-fold inter-individual variation respectively. It revealed that there were great inter-individual differences in CYP2A6 metabolic activity towards coumarin. 1.2 The effects of polymorphisms of ADH1 B rs1229984(G>A),ADH1 C rs698(A>G),ADH1 C rs2241894(A>G)、ALDH2 rs671(A>G),ALDH2 rs13306164(C>T) on the enzymes activityADH1B rs1229984(A> G) mutation frequency was 29.7%, ADH1 C rs698(A> G) mutation frequency was 10.5%, ADH1 C rs2241894(G> A) mutation frequency was 26.9%. Total ADH, ADHI, ADHII activity in AA, AG, GG three groups were no significant differences. The results showed that polymorphisms in the ADHs have no significant effect on the human liver ADH enzymes activity.ALDH2 rs671(A> G) mutation frequency was 87.3%, ALDH2 rs13306164(C> T) mutation frequency was 2.2%. For ALDH2 rs671(A> G) mutation, mutant GA total ALDH activity were lower than the wild-type; for ALDH2 rs13306164(C> T) mutation, mutant TC total ALDH activity and ALDH2 activity were lower than the wild type. The results showed that in normal liver specimens the polymorphisms in ALDH had a significant effect on the enzyme ALDH activity. 2 Liver cancer aldehyde dehydrogenases activity and gene polymorphisms 2.1 The total ADH,ADHI,ADHI and total ALDH、ALDH2 activityNormality test results show that the human liver homogenates total ADH, ADHI, ADHII and total ALDH, ALDH2 activity parameters were not normally distributed, so this experiment non-parametric test was used for statistical analysis. The median and range activity of total ADH,ADHI,ADHI and total ALDH、ALDH2 were 8.14(1.23~43.07),1.68(0.73~7.10),0.53(0.14~1.66),0.15(0.03~0.34),9.40(1.45~50.18) pmol·min-1·mg-1protein. The data displayed a 35.02、9.76、 11.51、12.93、34.71-fold inter-individual variation respectively. It revealed that there were great inter-individual differences in these enzyme activities. 2.2 The effects of polymorphisms of ADH1 B rs1229984(G>A),ADH1 C rs698(A>G),ADH1 C rs2241894(A>G)、ALDH2 rs671(A>G),ALDH2 rs13306164(C>T) on the enzymes activityADH1B rs1229984(A> G) mutation frequency was 38.1%, ADH1 C rs698(A> G) mutation frequency was 9.7%, ADH1 C rs2241894(G> A) mutation frequency was 28.4%. ADH1 B rs1229984 GG homozygote and heterozygote mutation ADH1 C rs698 AG and ADH1 C rs2241894 AG, AA mutant total ADH activity were significantly lower than the wild-type homozygotes.ALDH2 rs671(A> G) mutation frequency was 85.1%, ALDH2 rs13306164(C> T) mutation frequency was 5.2%. For ALDH2 rs671(A> G) mutation, mutant GA total ALDH activity were lower than the wild-type. The results showed that in normal liver specimens the polymorphisms in ALDH had a significant effect on the enzyme ALDH activity Conclusions1.There is great inter-individual difference in human liver total ADH, ADHI, ADHII and total ALDH, ALDH2 activity.2.For liver cancer specimens, the total ADH, ADHI, ADHII activity was significantly higher than the normal liver specimens, while there were no differences in ALDH2 activity. These changes may cause the increase of acetaldehyde, which further leads to liver cancer.3.There were no difference in ADH and ALDH gene polymorphism distribution between patients with normal liver function and liver cancer patients.4.For ALDH2 rs671(A>G) mutation, total ALDH and ALDH2 activity were significantly lower than the wild-type in both tissues; for ALDH2 rs13306164 C>T mutation, there were no significant effect on the enzyme activity; for ADH1 B rs1229984(A>G), ADH1Crs698(A>G), ADH1 C rs2241894(G>A) mutation the total ADH activity in liver cirrhosis with liver specimens was significantly lower than the wild-type activity, and in the normal liver specimens there were no significant effect on the function of normal liver samples. |
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