黄曲霉毒素B_1诱发大鼠肝癌过程中对CYP3A4活性的影响

目的探讨黄曲霉毒素B1（AFB1）诱发肝癌过程中对CYP3A4活性的影响。方法采用大鼠肝微粒体混合酶体外代谢体系,利用荧光定量法动态检测AFB1诱发肝癌过程中不同时期CYP3A4酶活性。结果 AFB1组CYP3A4含量从实验开始逐渐升高,至23周达顶峰,然后逐渐降低,到43周又升高,出现双波峰变化,阶段性比较差异有统计学意义（P=0.000）;对照组CYP3A4含量在整个实验过程中,除33周和63周出现明显降低外,其他各阶段变化不大;组间比较显示AFB1组在诱癌过程中有抑制CYP3A4的趋势,于63周抑制最明显,但尚未达统计学意义（P=0.638）;结论AFB1在诱癌过程中有抑制CYP3A4的趋势,可能是与早期癌变的细胞减少对特定基因毒性物质的活化有关。     

黄曲霉毒素B1诱发大鼠肝癌过程中对CYP3A4活性的影响

目的 探讨黄曲霉毒素B1(AFB1)诱发肝癌过程中对CYP3A4活性的影响.方法 采用大鼠肝微粒体混合酶体外代谢体系,利用荧光定量法动态检测AFB1诱发肝癌过程中不同时期CYP3A4酶活性.结果 AFB1组CYP3A4含量从实验开始逐渐升高,至23周达顶峰,然后逐渐降低,到43周又升高,出现双波峰变化,阶段性比较差异有统计学意义(P=0.000);对照组CYP3A4含量在整个实验过程中,除33周和63周出现明显降低外,其他各阶段变化不大;组间比较显示AFB1组在诱癌过程中有抑制CYP3A4的趋势,于63周抑制最明显,但尚未达统计学意义(P=0.638);结论AFB1在诱癌过程中有抑制CYP3A4的趋势,可能是与早期癌变的细胞减少对特定基因毒性物质的活化有关.     

CCT亚基γ基因在AFB1诱发大鼠肝癌过程中表达的变化

目的探讨AFB1诱发肝癌过程中CCT亚基γ基因蛋白的表达及其意义。方法45只Wistar大鼠随机分为AFB。组和对照组,AFB1组以200ug／kg体重腹腔注射AFB1,第13周、第33周和第53周对大鼠进行肝活检,第73周处死全部动物并取肝组织。用免疫组化法检测AFB1诱发肝癌过程中不同时期CCT亚基γ基因蛋白的表达情况。结果在第73周AFB1组CCT亚基γ基因蛋白表达水平显著高于对照组（P〈0．05）。同一组不同时段比较,随着诱癌的进行,CCT亚基γ基因蛋白表达上调,第73周AFB1组CCT亚基γ基因蛋白的表达水平不仅明显高于第13周,而且亦高于第33周（P〈0．01）。其余各组和同组不同时段之间CCT亚基γ基因蛋白的表达差异无统计学意义（P〉0．05）。AFB。组中发生肝癌的动物肝组织CCT亚基γ基因蛋白表达率为100％（10／10）,显著高于同期对照组CCT亚基γ基因蛋白的表达率30％（3／10）（P〈0．05）。结论AFB,诱癌过程中CCT亚基γ基因蛋白表达上调,CCT亚基γ基因蛋白有可能参与了肝细胞癌的发生和发展。     

大鼠肝癌形成过程MCM7基因动态变化

目的：4g讨MCM7基因在黄曲霉毒素B1（AFB1）实验诱发大鼠肝癌过程中的动态变化及意义。方法：将70只雄性4周龄Wistar大鼠随机分为AFB1组（50只）和对照组（20只）,AFB1组腹腔注射AFB1,对照纽则给予溶媒二甲基亚砜。在诱发肝癌过程中,分别于第13、33和53周对大鼠进行肝活检;实验至第73周处死全部大鼠取肝组织;采用实时荧光定量PCR、蛋白质印迹法和免疫组化方法检测肝组织中MCM7rnRNA和蛋白的表达情况。结果：AFB1组肝细胞癌发生率为44．1％（15／a4）,对照组为0（0／16）,AFB1组显著高于对照组,χ^2=8．093,P〈0．001。随着大鼠肝癌的形成,MCM7mRNA和蛋白的表达水平逐步升高,AFB1组第53和73周显著高于第13和33周,F值分别为55．632和207．253,P值均〈0．001;对照组各时间点的表达较低且变化不大。免疫组化结果显示,AFB1组第53周McM7蛋白阳性表达率为26．7％且以弱阳性为主,第73周MCM7蛋白阳性表达率为100％且多为强阳性,阳性表达率和表达强度均比其他时间点的高,两者比较差异均有统计学意义,P值均〈0．001。结论：MCM7表达水平随着肝癌的形成逐渐上升,且在肝癌组织中表达最高,MCM7有可能是参与肝癌形成的关键基因,但其详细机制有待进一步探讨。     

黄曲霉毒素诱导大鼠肝细胞癌变中JNK1的变化

目的 观察黄曲霉毒素诱导大鼠肝细胞癌变过程中JNK的变化,进一步了解JNK1信号转导通路在肝癌（Hepatocellular carcinoma,HCC）发生发展过程中的作用。方法 取雄性4周龄SD大鼠77只为研究对象,随机分为实验组66只,空白对照组11只。实验组应用黄曲霉毒素B1（Aflatoxin B1,AFB1）诱发大鼠肝细胞癌,空白对照组大鼠,按正常饲养方法饲养,两组大鼠分别于实验第12、20、36、46周进行肝组织活检,58周时处死取肝。所有标本均进行常规病理组织学检测,并应用RT-PCR、Western blot分别检测癌组织及肝组织JNK1 mRNA及JNK1活性蛋白质水平。结果 实验组大鼠46周时发现首例肝癌,存活至实验结束并发生肝癌的大鼠共24只,6只无任何肿瘤发生,空白对照组大鼠11例均未发生肿瘤。实验组及空白对照组大鼠均可检出不同程度的JNK1 mRNA表达,其中肝癌组织较癌旁组织及正常肝组织明显增强（P<0.05）。Western blot结果显示,实验组中无癌大鼠20周、出癌大鼠36周始即有不同程度p-JNK1活性表达,并随着AFB1作用时间延长阳性率明显增加,且无癌大鼠p-JNK1检出的时间较出癌大鼠早;半定量分析表明无癌大鼠肝组织p-JNK1活性较同期出癌大鼠肝组织及肝癌组织增强,在实验第46周及58周时尤其明显（P<0.01）。结论 在AFB1诱导肝细胞癌的过程中JNK信号通路处于激活状态,JNK信号通路的激活可能起到抑制HCC的作用,如JNK信号通路激活时间较早、强度较大可阻止HCC的发生。     

CCT亚基γ基因在AFB_1诱发大鼠肝癌过程中表达的变化

目的探讨AFB1诱发肝癌过程中CCT亚基γ基因蛋白的表达及其意义。方法 45只Wistar大鼠随机分为AFB1组和对照组,AFB1组以200μg/kg体重腹腔注射AFB1,第13周、第33周和第53周对大鼠进行肝活检,第73周处死全部动物并取肝组织。用免疫组化法检测AFB1诱发肝癌过程中不同时期CCT亚基γ基因蛋白的表达情况。结果在第73周AFB1组CCT亚基γ基因蛋白表达水平显著高于对照组(P<0.05)。同一组不同时段比较,随着诱癌的进行,CCT亚基γ基因蛋白表达上调,第73周AFB1组CCT亚基γ基因蛋白的表达水平不仅明显高于第13周,而且亦高于第33周(P<0.01)。其余各组和同组不同时段之间CCT亚基γ基因蛋白的表达差异无统计学意义(P>0.05)。AFB1组中发生肝癌的动物肝组织CCT亚基γ基因蛋白表达率为100%(10/10),显著高于同期对照组CCT亚基γ基因蛋白的表达率30%(3/10)(P<0.05)。结论 AFB1诱癌过程中CCT亚基γ基因蛋白表达上调,CCT亚基γ基因蛋白有可能参与了肝细胞癌的发生和发展。     

银杏叶对黄曲霉毒素B1诱发大鼠肝癌IGF-ⅡmRNA及蛋白表达影响的观察

目的：动态观察银杏叶提取物（EGb761）在抑制黄曲霉毒素B1（AFB1）诱发大鼠肝癌（HCO）过程中对肝组织相关基因IGF-ⅡmRNA及蛋白表达水平的影响,从分子生物学水平揭示银杏叶抗癌的机制及其效果。方法：将70只4周龄Wistar雄性大鼠随机分为AFB1组（25只）、AFB1＋EGb761组（25只）及对照组（20只）。在诱发大鼠HCC过程中,分别于第13周、33周及53周对大鼠进行肝组织活检;实验至第73周处死全部动物取肝组织。利用实时荧光定量PCR和蛋白质印迹法动态检测肝组织中IGF-ⅡmRNA及相应蛋白的表达情况。结果：AFB1组原发性HCC发生率为58．8％（10／17）;AFB1＋EGb761组发生率为29．4％（5／17）;对照组为0（o／16）。AFBl＋EGb761组HCC发生率显著低于AFB1组（P〈0．05）。AFBl＋EGb761组肝组织IGF_2mRNA及相应蛋白表达扮平在第53周及73周较AFB1组均显著下降,差异有统计学意义,P％0．05。结论：银杏叶提取物（EGb761）具有抑制AFBl致大鼠HCC的作用。其机制可能与调控肝细胞增殖基因IGF-ⅡmRNA及蛋白表达水平有关。     

银杏叶提取物对AFB1所致大鼠肝癌基因P16Ink4a mRNA及蛋白表达的影响

  目的  动态观察银杏叶提取物(ginkgo biloba extract, EGb761)在抑制黄曲霉毒素B1(AFB1)诱发大鼠肝癌过程中对肝组织相关基因P161nk4a mRNA表达水平的影响, 进一步从分子生物学水平揭示银杏叶提取物抗癌的机制及其效果。  方法  将70只4周龄Wistar雄性大鼠随机分为3组: AFB1组(25只), AFB1+EGb761组(25只)及对照组(20只)。在诱发肝癌过程中, 分别于第13、33及53周对大鼠进行肝组织活检; 至第73周处死全部动物取肝组织。应用实时荧光定量PCR和Westernblot技术动态检测肝组织中P16Ink4a mRNA及相应蛋白的表达情况。  结果  AFB1组原发性肝癌发生率为58.8%(10/17);AFB1+EGb761组为29.4%(5/17);对照组为0(0/16) AFB1+EGb761组肝癌发生率显著低于AFB1组(P < 0.05)。AFB1+EGb761组的肝组织P16Ink4amRNA及相应蛋白表达水平在第53周及第73周时均明显高于AFB1组, 差异有统计学意义(P < 0.05)。  结论  银杏叶提取物(EGb761)具有抑制AFB1致大鼠肝癌的作用。其机制可能与调控肝细胞抑癌基因P16Ink4a mRNA表达水平有关。      

银杏叶对黄曲霉毒素B1诱发大鼠肝癌IGF-Ⅱ mRNA及蛋白表达影响的观察

目的:动态观察银杏叶提取物(EGb761)在抑制黄曲霉毒素B1 (AFB1)诱发大鼠肝癌(HCC)过程中对肝组织相关基因IGF-ⅡmRNA及蛋白表达水平的影响,从分子生物学水平揭示银杏叶抗癌的机制及其效果.方法:将70只4周龄Wistar雄性大鼠随机分为AFB1组(25只)、AFB1+ EGb761组(25只)及对照组(20只).在诱发大鼠HCC过程中,分别于第13周、33周及53周对大鼠进行肝组织活检；实验至第73用处死全部动物取肝组织.利用实时荧光定量PCR和蛋白质印迹法动态检测肝组织中IGF-ⅡmRNA及相应蛋白的表达情况.结果:AFB1组原发性HCC发生率为58.8％(10/17)；AFB1+EGb761组发生率为29.4％(5/17)；对照组为0(0/16).AFB1+EGb761组HCC发生率显著低于AFB1组(P＜0.05).AFB1+ EGb761组肝组织IGF-2 mRNA及相应蛋白表达水平在第53周及73周较AFB1组均显著下降,差异有统计学意义,P＜o.05.结论:银杏叶提取物(EGb761)具有抑制AFB1致大鼠HCC的作用.其机制可能与调控肝细胞增殖基因IGF-ⅡmRNA及蛋白表达水平有关.     

用cDNA微阵列技术研究HBV X基因与AFB1对HBVx转基因小鼠药物代谢酶基因表达谱的影响

目的研究乙型肝炎病毒X基因和黄曲霉毒素诱发小鼠肝癌过程中药物代谢酶基因表达谱的变化,探讨两因素协同致肝癌的机制.方法用BiostarM-40s微阵列芯片比较研究HBVx组、AFB1组和(AFB1 HBVx)组的肝组织基因表达谱与对照组的差异.结果各实验组分别与对照组相比基因表达谱发生了明显的改变,各实验组上调与下调的基因数目分别为(AFB1 HBVx)组69项;AFB1组101项;HBVx组35项;其中与代谢酶相关的基因有18项表达发生改变,分别为(AFB1 HBVx)组13项(13/18,72%);HBVx组4项(4/18,22%);AFB1组8项(8/18,44%).结论小鼠受到HBV X基因和AFB1双重攻击后,其体内的GST、EPHX和UDPGT等药物代谢酶基因表达水平明显低于HBVx组和AFB1组.HBV与AFB1协同致癌的分子机制很可能与两者引起药物代谢酶基因表达水平下调有关.     

Effects of Ginkgo biloba extract on expression of biomarkers during aflatoxin B1-induced hepatocarcinogenesis in Wistar rats

Objective

The aim of this study was to study the effect of Ginkgo biloba extract (EGb761) on metabolism of aflatoxin B₁ (AFB₁) in Wistar rats.

Methods

Seventy one Wistar rats were assigned at random to groups A, B and C. Rats in groups A, B were injected with AFB₁ (intraperitoneal, 100–200 μg/kg body weight, 1–3 times/week). Group C was normal control. Rats in group B were fed in food with EGb761, while rats in groups A, C were given normal food. Blood samples were collected and liver biopsies were performed on the 14th, 28th and 42nd week. All the rats were sacrificed on the 64th week. The incidence of hepatocarcinoma was investigated. The hepatic phase I drug-metabolizing enzyme Cytochrome-P450 (CYP450) and phase II metabolizing enzyme glutathione S-transferase (GST) were analyzed with spectrometry. Serum AFB₁-lysine adduct levels were assessed with high performance liquid chromatography (HPLC). The expression of 8-hydroxydeoxyguanosine (8-OHdG) was measured with immunohistochemistry.

Results

The incidence of hepatocellular carcinoma (HCC) in group B was significantly lower than that in group A (26.92% vs 76.00%, P < 0.001). No HCC developed in group C. EGb761 showed no effects on the activities of CYP450 and GST in rat liver tissues. The level of AFB1-lysine adduct reached the peak (4356.01 pg/mg albumin) at the 14th week in group A. EGb761 significantly inhibited the formation of AFB1-lysine adduct in serum by 13.07% at the 14th week (P = 0.033), and 73.63% at the 42nd week (P = 0.002). The expression of 8-OHdG protein in rat liver tissues in group B was significantly lower than that in group A at the 28th, 42nd, and 64th week (P < 0.05).

Conclusion

The main mechanism underlying the effect of EGb761 in blocking hepatocarcinogenesis induced by AFB1 may not be fully attributable to its influence on the activity of liver phase I and phase II metabolizing enzymes. EGb761 inhibits the production of AFB₁-lysine adducts, decreases the expression of 8-OHdG protein, and finally alleviates the DNA oxidative injury, which may be one of the mechanisms for the effects of EGb761 in inhibiting or delaying AFB1-induced hepatocarcinogenesis.     

Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract

The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1 in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1-8,9-epoxide and AFM1-8,9-epoxide at both low (15 microM) and high (150 microM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an approximately 800-fold difference in LC50 values of AFB1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 microM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.     

Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry.

Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancing effect of ethanol on aflatoxin B1-induced hepatocarcinogenesis in male ACI/N rats

The modifying effect of ethanol (EtOH) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis was examined in male ACI/N rats by chronic treatment at the post-initiation phase. Rats received an ip injection of AFB1 (1.5 mg/kg) twice a week for 10 weeks (a total of 20 doses). Following a week of acclimation, they were given 10% EtOH as drinking water for 56 weeks. The effect of EtOH on the hepatocarcinogenesis was evaluated in terms of the incidence of altered hepatocellular foci and neoplasms at the end of the experiment. Exposure to AFB1 alone induced a substantial number of altered foci (6.98 iron-excluding foci/cm2) in rats. The number of altered liver cell foci in rats receiving AFB1 followed by EtOH was significantly increased (26.39 iron-excluding foci/cm2). In the rats given EtOH after AFB1, the total area and mean diameter of both iron-excluding foci and altered foci identified in hematoxylin and eosin-stained sections were significantly higher than in the rats exposed to AFB1 alone. The incidence of liver cell tumors of the group given AFB1 and EtOH (3/15, 20%) was higher than that of the group treated with AFB1 alone (0/14, 0%). Treatment with EtOH alone for 56 weeks did not induce either. These results indicate an enhancing effect of EtOH on AFB1-induced hepatocarcinogenesis when it is given in the promotion phase.