乙醇相关氧化应激在广西原发性肝癌发病及经皮无水酒精注射治疗反应性中的作用
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摘要
目的:分析乙醇相关的氧化应激反应及调节机制,探讨其与广西原发性肝癌发病及乙醇治疗肝癌反应性的关系。
方法:
(1)采用多聚酶链反应-限制性片段长度多态性(PCR-RFLP)技术,对广西地区300例原发性肝癌和292例健康志愿者行病例-对照分析,检测乙醇代谢酶ALDH2和CYP2E1基因型,分析基因—环境、基因—基因相互作用与原发性肝癌发生的关系。
(2)采取20例肝癌病人和10例健康人外周血,分选的淋巴细胞作为研究对象,流式细胞术检测不同浓度乙醇暴露下的氧化性DNA损伤标志物8-oxoG水平,同时测定细胞内羟自由基(OH~-)含量、活性氧(ROS)含量、丙二醛(MDA)及超氧化物岐化酶(SOD)水平,了解乙醇暴露下淋巴细胞的氧自由基含量、脂质过氧化损伤水平和其抗氧化能力。同时应用RT-PCR技术检测乙醇暴露下淋巴细胞的乙醇代谢酶和DNA损伤修复酶基因ALDH2、CYP2E1、hOGG1、APE的转录表达,并对其基因进行分型。采用基因组学与毒理学相结合的方法,结合基因型与体外实验功能表型验证影响乙醇相关氧化性损伤的功能性基因型。
(3)选取20例行无水酒精注射(PEI)治疗的肝占位病人作为研究对象,测定PEI术前及术后的丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,并采用PCR方法检测乙醇代谢酶、DNA损伤修复酶ALDH2、CYP2E1、hOGG1和APE基因型,结合基因型与体内实验功能表型验证影响PEI术后转氨酶的功能性基因型。
结果:
(1)肝癌组和对照组中ALDH2变异基因型和CYP2E1变异基因型携带者分别占50.3%、48.0%和32.3%和32.9%(P＞0.05)。饮酒频度每周≥3次且携带变异ALDH2~*2和CYP2E1c2基因型者发生肝癌的危险度分别是饮酒频度每周＜3次且携带野生ALDH2~*1和CYP2E1c1基因型者的3.334倍(95%CI=1.746～6.406)和1.803倍(95%CI=0.974～3.336),同时携带两变异基因型者患肝癌风险为1.200倍(95%CI=0.730～1.972),且饮酒增加两变异基因型携带者的肝癌发病风险(OR=1.816,95%CI=0.985～3.348)。HBsAg携带者明显增加肝癌患病风险(OR=30.800,95%CI=14.480～65.513),HbsAg阳性且饮酒频度每周＜3次和饮酒频度每周≥3次者患肝癌危险度分别是HBsAg阴性且不饮酒者的52.617倍(95%CI=21.122～131.070)和72.893倍(95%CI=25.197～210.872)。
(2)肝癌组淋巴细胞8-oxoG水平均高于对照组;乙醇干预后,淋巴细胞损伤8-oxoG水平均高于空白组(P＜0.05)。淋巴细胞上清液OH~-含量,随着乙醇浓度的升高而逐渐增加,各剂量组与空白组相比,差异有非常显著性(P＜0.01)。偏相关分析显示8-oxoG水平与OH~-含量呈显著正相关(r=0.9729,P=0.005)。携带野生ALDH2 484Glu或hOGG1 326Ser基因型个体的淋巴细胞DNA损伤高于携带变异ALDH2 484Lys或hOGG1 326Cys基因型的个体,两者之间的差异有统计学意义(P＜0.05)。
(3)PEI术后肝转氨酶指标均升高,其中以第一次PEI术后转氨酶升高最为显著(P＜0.05),第三次PEI以后的转氨酶改变逐渐趋缓,但仍高于术前。前三次PEI术后的ALT,ALDH2~*1基因型组显著高于ALDH2~*2基因型组(P＜0.01),第一和第三次PEI术后的AST,ALDH2~*1基因型组高于ALDH2~*2基因型组(P＜0.05和P＜0.01)。CYP2E1c2基因型组,PEI术后的转氨酶均高于c1基因型组,尤其第四次和第五次PEI术后的ALT和第二次PEI术后的AST显著升高(与c1基因型组相比,P＜0.05)。
结论:
(1)单一易感基因ALDH2或CYP2E1的基因型与肝癌易感性无关;高频饮酒(饮酒频度每周≥3次)且携带变异ALDH2 484Lys或CYP2E1 c2基因型者患肝癌的风险增加,且两变异基因的单倍体者增加肝癌发病风险。HBV感染是肝癌发病的最主要因素之一,饮酒显著增加HBsAg携带者的肝癌风险。提示对于变异基因ALDH2~*2或和CYP2E1c2携带者而言,高频饮酒是一个重要的危险因子;乙醇在增加肝癌发病风险的过程中存在基因—环境和基因—基因相互作用。
(2)低浓度乙醇暴露后的淋巴细胞损伤主要由乙醇诱导产生过多的OH~-所致。低浓度乙醇暴露后的淋巴细胞8-oxoG水平与代谢酶ALDH2和修复酶hOGG1基因型相关。携带ALDH2 484Glu或hOGG1 326Ser基因型者,乙醇暴露后的淋巴细胞DNA损伤程度较重。
(3)PEI术后转氨酶呈先升高再降低的变化趋势,以第一次PEI术后转氨酶升高最为显著。野生型ALDH2 484Glu或突变型CYP2E1c2基因携带者PEI术后的肝损伤程度较重。
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.
方法:
(1)采用多聚酶链反应-限制性片段长度多态性(PCR-RFLP)技术,对广西地区300例原发性肝癌和292例健康志愿者行病例-对照分析,检测乙醇代谢酶ALDH2和CYP2E1基因型,分析基因—环境、基因—基因相互作用与原发性肝癌发生的关系。
(2)采取20例肝癌病人和10例健康人外周血,分选的淋巴细胞作为研究对象,流式细胞术检测不同浓度乙醇暴露下的氧化性DNA损伤标志物8-oxoG水平,同时测定细胞内羟自由基(OH~-)含量、活性氧(ROS)含量、丙二醛(MDA)及超氧化物岐化酶(SOD)水平,了解乙醇暴露下淋巴细胞的氧自由基含量、脂质过氧化损伤水平和其抗氧化能力。同时应用RT-PCR技术检测乙醇暴露下淋巴细胞的乙醇代谢酶和DNA损伤修复酶基因ALDH2、CYP2E1、hOGG1、APE的转录表达,并对其基因进行分型。采用基因组学与毒理学相结合的方法,结合基因型与体外实验功能表型验证影响乙醇相关氧化性损伤的功能性基因型。
(3)选取20例行无水酒精注射(PEI)治疗的肝占位病人作为研究对象,测定PEI术前及术后的丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,并采用PCR方法检测乙醇代谢酶、DNA损伤修复酶ALDH2、CYP2E1、hOGG1和APE基因型,结合基因型与体内实验功能表型验证影响PEI术后转氨酶的功能性基因型。
结果:
(1)肝癌组和对照组中ALDH2变异基因型和CYP2E1变异基因型携带者分别占50.3%、48.0%和32.3%和32.9%(P＞0.05)。饮酒频度每周≥3次且携带变异ALDH2~*2和CYP2E1c2基因型者发生肝癌的危险度分别是饮酒频度每周＜3次且携带野生ALDH2~*1和CYP2E1c1基因型者的3.334倍(95%CI=1.746～6.406)和1.803倍(95%CI=0.974～3.336),同时携带两变异基因型者患肝癌风险为1.200倍(95%CI=0.730～1.972),且饮酒增加两变异基因型携带者的肝癌发病风险(OR=1.816,95%CI=0.985～3.348)。HBsAg携带者明显增加肝癌患病风险(OR=30.800,95%CI=14.480～65.513),HbsAg阳性且饮酒频度每周＜3次和饮酒频度每周≥3次者患肝癌危险度分别是HBsAg阴性且不饮酒者的52.617倍(95%CI=21.122～131.070)和72.893倍(95%CI=25.197～210.872)。
(2)肝癌组淋巴细胞8-oxoG水平均高于对照组;乙醇干预后,淋巴细胞损伤8-oxoG水平均高于空白组(P＜0.05)。淋巴细胞上清液OH~-含量,随着乙醇浓度的升高而逐渐增加,各剂量组与空白组相比,差异有非常显著性(P＜0.01)。偏相关分析显示8-oxoG水平与OH~-含量呈显著正相关(r=0.9729,P=0.005)。携带野生ALDH2 484Glu或hOGG1 326Ser基因型个体的淋巴细胞DNA损伤高于携带变异ALDH2 484Lys或hOGG1 326Cys基因型的个体,两者之间的差异有统计学意义(P＜0.05)。
(3)PEI术后肝转氨酶指标均升高,其中以第一次PEI术后转氨酶升高最为显著(P＜0.05),第三次PEI以后的转氨酶改变逐渐趋缓,但仍高于术前。前三次PEI术后的ALT,ALDH2~*1基因型组显著高于ALDH2~*2基因型组(P＜0.01),第一和第三次PEI术后的AST,ALDH2~*1基因型组高于ALDH2~*2基因型组(P＜0.05和P＜0.01)。CYP2E1c2基因型组,PEI术后的转氨酶均高于c1基因型组,尤其第四次和第五次PEI术后的ALT和第二次PEI术后的AST显著升高(与c1基因型组相比,P＜0.05)。
结论:
(1)单一易感基因ALDH2或CYP2E1的基因型与肝癌易感性无关;高频饮酒(饮酒频度每周≥3次)且携带变异ALDH2 484Lys或CYP2E1 c2基因型者患肝癌的风险增加,且两变异基因的单倍体者增加肝癌发病风险。HBV感染是肝癌发病的最主要因素之一,饮酒显著增加HBsAg携带者的肝癌风险。提示对于变异基因ALDH2~*2或和CYP2E1c2携带者而言,高频饮酒是一个重要的危险因子;乙醇在增加肝癌发病风险的过程中存在基因—环境和基因—基因相互作用。
(2)低浓度乙醇暴露后的淋巴细胞损伤主要由乙醇诱导产生过多的OH~-所致。低浓度乙醇暴露后的淋巴细胞8-oxoG水平与代谢酶ALDH2和修复酶hOGG1基因型相关。携带ALDH2 484Glu或hOGG1 326Ser基因型者,乙醇暴露后的淋巴细胞DNA损伤程度较重。
(3)PEI术后转氨酶呈先升高再降低的变化趋势,以第一次PEI术后转氨酶升高最为显著。野生型ALDH2 484Glu或突变型CYP2E1c2基因携带者PEI术后的肝损伤程度较重。
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.
引文
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