The Role Of Alcohol-related Oxidative Stress In The Development Of Primary Hepatocellular Carcinoma And The Response To Percutaneous Ethanol Injection In Guangxi, PR China

Objective:To study the role of alcohol-related oxidative stress in the development of primary hepatocellular carcinoma(HCC)in Guangxi,PR China, and further explore the role of oxidative stress in modulating individual response to percutaneous ethanol injection(PEI).Methods:(1)Three hundred HCCs and 292 controls were genotyped for the aldehyde dehydrogenase-2(ALDH2)and CYP2E1 polymorphisms using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)method.(2)Peripheral blood lymphocytes were collected from 20 HCC patients and 10 healthy people.Oxidative DNA damage levels in lymphocytes with regard to gradient doses of ethanol were quantified by flow cytometry.Intracellular superoxide dismutase(SOD),lipid peroxidation products-malondialdehyde (MDA),hydroxyl radicals(OH)and reactive oxygen species(ROS)were also determined to evaluate the antioxidant capacity of ethanol,the level of lipid peroxidation and the oxidative effects in cultured cells.In addition,the expression levels of ethanol metabolism and DNA repair genes (ALDH2,CYP2E1,hOGG1 and APE)were measured by RT-PCR assays,and the genotypes of ALDH2,CYP2E1,hOGG1 and APE were detected by PCR-based assays.(3)Twenty patients with focal hepatic lesions received percutaneous ethanol injection(PEI).Serum ALT and AST were monitored pre and post PEI,and the genotypes of ALDH2,CYP2E1,hOGG1 and APE genes were detected using PCR-based assays.Results:(1)The frequencies of ALDH2 and CYP2E1 variant genotypes in cases and controls were 50.3%,48.0%and 32.3%,32.9%,respectively.There was no significant difference of ALDH2 and CYP2E1 genotypes distribution between cases and controls(P＞0.05).The risk for liver cancer was 3.334 times higher in alcoholics(≥3 times drinking per week)with ALDH2~*2 genotype than that that in cases carrying ALDH2~*1 genotype while drinking less than 3 times per week (95%CI=1.746～6.406),and the risk for liver cancer was 1.803 times higher in alcoholics(≥3 times drinking per week)with CYP2E1c2 genotype than that in cases carrying CYP2E1c1 genotype while drinking less than 3 times per week (95%CI=0.974～3.336).Haplatype of the two variant genotypes increased liver cancer risk to 1.200 folds(95%CI=0.730～1.972),and interaction between drinking and genotypes increases risk of liver cancer to 1.816 folds (95%CI=0.985～3.348).The HBsAg carriers had significant risk for liver cancer(OR=30.800,95%CI=14.480～65.513),the risk for liver cancer was 52.617 times(95%CI=21.122～131.070)and 72.893 times(95%CI= 25.197～210.872),respectively,higher in drinking less than 3 times per week and alcoholics(≥3 times drinking per week)with HBsAg positive than that in cases while not drinking with HBsAg negative.(2)After ethanol incubation,the 8-oxoG levels in lymphocytes(positive rate and the fluorescence intensity)were higher than that in controls,and the 8-oxoG levels in lymphocytes in HCC cases were higher than that in controls(P＜0.05). OH~- content in the supernatant increased with the ethanol concentration in a dose-dependent manner.Also,OH~- levels were associated with 8-oxoG levels in lymphocytes(r=0.9729,P=0.005).Individuals carrying ALDH2 484Glu and hOGG1 326Ser genotypes had higher DNA damage levels than those carrying ALDH2 484Lys and hOGG1 326Cys genotypes(P＜0.05). (3)Serum levels of ALT and AST in patients significantly increased after PEI intervention(P＜0.01)but the changes attenuated after repeated treatment.The serum levels of ALT in ALDH2 484Glu carriers were higher(P＜0.01)than that in the ALDH2 484Lys carriers after the first three sequential PEI treatment.The serum levels of AST in ALDH2 484Glu carriers were higher than that in the ALDH2 484Lys carriers after the first and third time PEI(P＜0.05 and P＜0.01). The serum levels of ALT in CYP2E1c2 carriers was higher(P＜0.05)than that in the CYP2E1c1 carriers after the forth and fifth PEI treatment.The serum levels of AST in CYP2E1c2 carriers were higher than that in CYP2E1c1 carriers after the second time PEI treatment(P＜0.05).Conclusion:(1)ALDH2 or CYP2E1 genotypes alone render no significant risk for HCC, while frequent alcoholic consumption together with ALDH2 or CYP2E1 variant genotypes are assocoated with risk of hepatocarcinogenesis,HBV infection is one of the mainly factors for liver cancer,alcohol drinking increase the HBsAg carriers risk for liver cancer,suggesting a gene-environment interaction in increasing risk for HCC among Guangxi residents.(2)Upon low-concentration exposure to ethanol,damage to lymphocytes is induced mainly by excessive OH~-.In addition,after low concentration ethanol exposure,8-oxoG levels in lymphocytes are associated with ALDH2 and hOGG1 genotypes.ALDH2 484Glu or hOGG1 326Ser carriers have higher levels of DNA damage in their lymphocytes.(3)There is significant increase of ALT and AST levels in patients receiving the first dosage of PEI.ALDH2 484Glu and CYP2E1 c2 carriers may suffer severe liver injury after PEI treatment.