Minimal dose and time protection by lindane (gamma-isomer of 1,2,3,4,5,6 hexachlorocyclohexane) against liver tumors induced by aflatoxin B1

This study was carried out in order to investigate the minimal exposure to lindane (LD, 99.72% gamma isomer of 1,2,3,4,5,6 hexachlorocyclohexane), a chlorinated hydrocarbon insecticide, required to protect against liver tumor induced by aflatoxin B1 (AFB1). Materials fed to Buffalo strain rats were as follows: LD 100 ppm; AFB1 1 ppm, LD 100 ppm plus AFB1 1 ppm; and control basal diet. The experimental animals were clinically observed and then serially killed at 1, 3, 5, 10, 15 and 82 weeks. Concurrent administration of LD with AFB1 to rats for more than 3 weeks totally inhibited the incidence of AFB1-induced hepatocellular carcinomas by week 82. Only 1 of 20 rats (5%) fed the same regimen for 1 week developed liver tumors. Animals given 1 ppm AFB1 singly for 15 weeks had a high liver tumor incidence (31.5%). No animals developed liver tumors in LD-treated and control groups. LD may inhibit AFB1-induced liver tumors by stimulating hepatic metabolism and excretion of AFB1 so that less carcinogen is available to liver tissue.     

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.     

A single dose-response effect of aflatoxin B1 on rapid liver cancer induction in two strains of rats

To investigate rapid liver cancer induction in rats by aflatoxin B1 (AFB1), different single oral doses of AFB1 were given to 3 groups of 1-year-old Buffalo and Wistar rats. The animals were treated once and all survivors were killed 6 weeks later. Control animals received an equal volume of solvent (DMSO), and both groups of animals were maintained under identical conditions throughout the period of experiment. The survival rates were 40% with low and medium doses in AFB1-treated Buffalo and Wistar rats, and 0% in the high-dose Buffalo rats. Slight ante-mortem elevations in serum concentrations of glutamic pyruvic transaminase (SGOT) and glutamic oxaloacetic transaminase (SGPT) were indicative of the persistent damage effect of AFB1 at week 6. Total protein and albumin concentrations were not altered. The percent incidence of altered cell foci (areas) and neoplastic nodules was higher in Wistar than in Buffalo rats given a similar low dose. Various stages of well differentiated hepatocellular carcinomas (0.1-0.2 cm in diameter) developed in 3 of 8 Wistar rats. It thus appears that Wistar rats are more susceptible to hepatocarcinogenesis following a single oral dose of AFB1 than Buffalo rats.     

Suppression of aflatoxin B1-induced hepatotoxic lesions by crocetin (a natural carotenoid)

The suppressive effects of crocetin (a natural carotenoid) on the hepatotoxic lesions induced by aflatoxin B1 (AFB1) were investigated in male Wistar rats. Rats were divided into five groups: groups I and II served as normal and solvent control respectively. Group III was given AFB1 (25 micrograms/day/rat) alone; group IV was given crocetin (0.1 mg/day/rat) alone; and group V received both AFB1 and crocetin. Rats received AFB1 and crocetin for 9 and 10 weeks respectively, and were maintained on basal diet for 35 weeks. At the end of the experiment (week 45), the incidence of liver lesions in rats of group V was significantly reduced by approximately 40% compared with group III. There were no liver lesions in rats of groups I, II and IV. A significant protective effect of crocetin on AFB1 hepatotoxicity was shown, as manifested by reduced effects on the activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase (P less than 0.01-0.001). From our previous results and present data, we suggest that the suppression of crocetin on AFB1 hepatotoxicity in the rats might be due to the defense mechanisms of hepatic tissues that elevated the GSH S-transferase activity and decreased the formation of hepatic AFB1-DNA adducts.     

Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver

The involvement of tumor promotion in the hepatocarcinogenic action of peroxisome proliferators has not been generally accepted. We studied the effect of nafenopin (NAF) as a model compound in a two-stage initiation-promotion protocol. Carcinogenesis was initiated by a single dose of aflatoxin B1 (AFB1) in female (AFB1, 5 mg/kg) and male (AFB1, 2 mg/kg) Wistar rats. After recovery NAF was fed via the diet, providing a daily dose of 100 mg/kg body weight. Phenobarbital (PB) (50 mg/kg body weight) was fed to female rats as a positive control. The following results were obtained. (a) At weeks 40, 55, 59, and 70, significantly more and larger liver tumors were present in AFB1-NAF-treated rats than in rats receiving either compound alone, and the effect of the combined treatment was clearly more than additive, in three independent experiments including both sexes. This suggests tumor promotion by NAF. Male rats responded more strongly than females. Similarly, PB enhanced the yield of liver tumors. Histologically, tumors were hepatocellular adenoma or carcinoma. In group AFB1-PB the majority consisted of eosinophilic and glycogenstoring cells. However, adenoma and carcinoma of groups AFB1-NAF and O-NAF consisted of weakly basophilic cells. (b) Phenotypically altered foci were evaluated in hematoxylin- and eosin-stained liver sections from the female rats. NAF treatment after AFB1 had little effect on number and size of eosinophilic-clear cell foci and decreased the number of trigroid foci. However, it led to a dramatic increase (20-fold after 70 weeks of NAF treatment) in number and size of foci of a special phenotype that was extremely rare after AFB1 alone and virtually absent in group AFB1-PB. Hepatocytes in these foci are characterized by weak diffuse basophilia and some eosinophilia, similar to the phenotype in adenoma and carcinoma, and by absence of gamma-glutamyltranspeptidase (GGT) expression. Based on these findings, we propose the hypothesis that NAF promotes the development of liver tumors via a mechanism involving amplification of a specific subtype of altered hepatic foci.     

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。     

Aflatoxin-B exposure does not lead to p53 mutations but results in enhanced liver cancer of Hupki (human p53 knock-in) mice

Hepatocellular carcinoma (HCC) is a common human malignancy that is often associated with risk factors such as aflatoxin-B1 (AFB1) exposure and Hepatitis-B virus infection in developing countries. There is a strong correlation between these risk factors and mutation of the tumor-suppressor gene p53 at codon 249. In vitro experiments have also shown that treatment of human liver cells with AFB1 results in p53 mutations. A tumor-promoting role for mutant p53 was demonstrated using transgenic mice models, in which HCC development was accelerated upon AFB1-exposure. However, wild-type mice in which AFB1 alone was used to induce liver cancers have failed to recapitulate p53 mutations, raising the possibility that mouse DNA context may not be appropriate for the generation of AFB1-induced p53 mutations. We have now tested this hypothesis using the Hupki mice (human p53 knock-in) in which the mouse DNA-binding domain has been replaced by the homologous human p53 segment. Mice were followed for 80 weeks after AFB1 injection for survival and HCC formation. Hupki mice were found to be more susceptible to AFB1 than wild-type mice. Moreover, only 19% of wild-type mice developed HCCs compared to 44% in Hupki mice. However, none of the liver tumors and normal tissues from Hupki mice contained any mutations in the DNA-binding domain of p53. These findings suggest that the human DNA context of the p53 gene alone may not be the sole determinant of AFB1-induced mutagenesis. Furthermore, humanized p53 appears not to be as effective as murine p53 in the mouse cellular environment in preventing malignant transformation.     

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。     

Enhancement of GST-P-positive liver cell foci development by nivalenol, a trichothecene mycotoxin.

In order to elucidate whether T-2 toxin (T-2) and nivalenol (NIV), the naturally occurring trichothecene mycotoxins in food and feed, are carcinogenic or possess an ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity, a medium-term liver bioassay was carried out. F344 male rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg), and then fed the test trichothecenes in diet (2 and 5 p.p.m. T-2 or 6 p.p.m. NIV) for 6 weeks beginning 2 weeks after the injection. Some control groups received DEN alone. For synergism between AFB1 and the trichothecenes, DEN-initiated rats as above were given a single i.p. injection of AFB1 (0.5 mg/kg) 2 weeks later and were fed a NIV-containing diet (6 p.p.m.) for 6 weeks. The other control group received the vehicle alone. Control rats not initiated with DEN were also treated with AFB1, NIV or T-2 alone as above. All rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and killed at week 8, and liver sections were analyzed by glutathione S-transferase placental form (GST-P) expression. In rats that did not receive DEN, AFB1 alone enhanced both the numbers and areas of GST-P-positive foci as reported earlier, while NIV or T-2 alone induced no marked changes. In rats initiated with DEN, AFB1 caused a marked expression of GST-P, and thus the hepatocarcinogenicity of AFB1 was reconfirmed. The expression of GST-P foci in rats fed T-2 or NIV was found to be at background level, indicating that the hepatocarcinogenicity was not predicted for the trichothecene mycotoxins such as T-2 and NIV by this medium-term bioassay system. In the group initiated by DEN followed by AFB1, on the other hand, an elevation of both the numbers and areas of GST-P-positive foci was observed by the subsequent feeding of rats with NIV, and this elevation was statistically significant from the sum totals of individual data of AFB1 or NIV alone. From this evidence, it is predicted that NIV causes an enhancing effect on AFB1-induced hepatocarcinogenesis.     

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。      

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.     

Inhibitory effect of the non-steroidal anti-inflammatory drug, indomethacin on the naturally occurring carcinogen, 1-hydroxyanthraquinone in male ACI/N rats

The effect of the non-steroidal anti-inflammatory drug indomethacin on 1-hydroxyanthraquinone (1-HA)-induced carcinogenesis was investigated in male ACI/N rats. Animals were fed the diet containing 1.5% 1-HA and simultaneously given indomethacin solution (16 p.p.m.) as drinking water for 48 weeks. The incidences of large bowel neoplasms (adenomas and adenocarcinomas) and forestomach (papillomas) in rats given 1-HA and indomethacin (large intestinal tumors: 0/14, 0%; forestomach tumors: 2/14, 14%) were significantly lower than those in rats given 1-HA alone (large intestinal tumors: 12/27, 44%; forestomach tumors: 14/27, 52%) (P = 0.002 and P = 0.01 respectively). Liver cell adenomas were developed in a rat given 1-HA and no liver tumors in rats treated with 1-HA and indomethacin. Altered liver cell foci were present in rats given 1-HA alone (18/27, 67%) and those given 1-HA and indomethacin (8/14, 57%), but no significant difference in the incidence between the two groups was found. Untreated animals and rats given indomethacin alone had no neoplasms in the large bowel, forestomach and liver. Thus, the non-steroidal anti-inflammatory drug indomethacin significantly inhibited carcinogenesis induced by the naturally occurring carcinogen, 1-HA.     

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 AFB, 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.     

Aflatoxin-DNA adduct formation in chronically dosed rats fed a choline-deficient diet

Nutritional modulation of male Fischer rats by a choline-deficient/methionine-low diet dramatically increases hepatocarcinogenesis and reduces time to first tumors induced by aflatoxin B1 (AFB1). The effect of this diet on hepatic aflatoxin-DNA adduct burden in male Fischer rats dosed with a carcinogenic regimen of AFB1 was examined in this study. After 3 weeks of ingestion of a choline-deficient/methionine-low diet or control semi-purified diet, rats were administered a carcinogenic regimen of 25 micrograms [3H]AFB1 for 5 days a week over 2 weeks. Six choline-deficient and four control diet rats were killed 2 h after each dose, and liver DNA isolated. In addition, hepatic DNA was isolated from animals 1, 2, 3, and 11 days after the last [3H]AFB1 administration. At all time points HPLC analysis of aflatoxin-DNA adducts was performed to confirm radiometric determinations of DNA binding levels. No significant quantitative differences in AFB1-DNA adduct formation between the dietary groups were observed following the first exposure to [3H]AFB1; however, total aflatoxin-DNA adduct levels in the choline-deficient animals were significantly increased during the multiple dose schedule. When total aflatoxin-DNA adduct levels were integrated over the 10 day dose period, a 41% increase in adduct burden was determined for the choline-deficient animals. While this increase in DNA damage is consistent with the hypothesis that DNA damage is related to tumor outcome, the biochemical basis for this effect still needs to be elucidated.     

Chemopreventive Potential of Neem Flowers on Carcinogen-induced Rat Mammary and Liver Carcinogenesis

We have previously reported that dietary neem flowers (Azadirachta indica A. Juss var. siamensis Valeton) caused ‍a marked increase in glutathione S-transferase (GST) activity in the liver, while resulting in a significant reduction ‍in the activities of some hepatic P450-dependent monooxygenases. These results strongly indicate that neem flowers ‍may have chemopreventive potential. In the present study, we examined the inhibitory effects of neem flowers on ‍9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague Dawley rats ‍and on aflatoxin B ‍1 ‍(AFB1)-induced hepatocarcinogenesis in male Wistar rats. Young animals were fed with AIN-76 ‍purified diets containing either 10-12.5% ground freeze-dried neem flowers for 1 week prior to, during, and for 1 ‍week after the administration of each carcinogen. Interestingly, it was found that neem flowers resulted in a marked ‍reduction of the incidence of mammary gland (about 35.2%) and liver tumors (61.7% and 80.1% for benign and ‍malignant tumors, respectively). Furthermore, the multiplicity of tumors per rats was also lower in the neem flower ‍groups, i.e. those for mammary gland tumors and benign and malignant liver tumors were reduced to 44.0%, ‍87.9% and 88.9%, respectively. These results clearly demonstrated that neem flowers contain some chemopreventive ‍agents capable of inhibiting AFB1 and DMBA induced liver and mammary gland carcinogenesis in rats.     

大鼠实验性肝癌发生过程中存活素基因表达的动态观察

目的动态观察大鼠实验性肝细胞肝癌(HCC)不同发生发展阶段中存活素(survivin)基因的表达状态。方法采用免疫组织化学法和逆转录-聚合酶链反应(RT-PCR)法,检测大鼠HCC不同时期survivin的表达。结果黄曲霉毒素(AFB1)诱发大鼠HCC形成的最早时间在第46周,46周后,实验组存活的大鼠HCC发生率为54.9%(28/51),58周存活的大鼠HCC发生率为64.9%(24/37)。实验组HCC、癌旁肝组织survivin蛋白阳性率分别为41.7%和54.2%,两者差异无统计学意义(P>0.05)。而相应的大鼠,在46周之前及无肿瘤形成的大鼠和正常对照组无survivin蛋白表达。58周时,大鼠HCC中survivin mRNA的表达水平明显高于同期无肿瘤形成大鼠和正常对照肝组织(P均<0.01);相应癌旁肝组织亦明显高于同期无肿瘤形成大鼠和对照肝组织(P均<0.01);HCC与癌前活检肝组织比较,差异有统计学意义(P<0.01)。12、20、36和46周时,有肿瘤形成大鼠活检肝组织与正常对照组大鼠同期活检肝组织比较,survivin mRNA表达差异均有统计学意义(P均< 0.01)。结论survivin基因在HCC组织中高表达,提示该基因对HCC的发生发展起重要作用,检测HCC组织中survivin基因的表达,可能对HCC诊断有参考价值。     

广西原发性肝细胞癌黄曲霉毒素B_1-DNA加合物表达及p53第249密码子突变分析

目的通过研究广西地区肝癌患者中黄曲霉毒素B1（Aflatoxin B1,AFB1）-DNA加合物的表达和p53第249密码子突变情况及其关系,探讨AFB1长期暴露与人群原发性肝细胞癌（hepatocellular carcinoma,HCC）发生的关系。方法实验组为广西医科大学第一附属医院肝胆外科收治的63例HCC患者行根治性手术切除的肝肿瘤组织。按肿瘤大小分为小肝癌组（≤5cm）和大肝癌组（〉5cm）,同时小肝癌组包括两个亚组Ⅰ组（≤3cm）和Ⅱ组（〉3cm）。另取10例来自肝移植供肝及肝外伤切除的正常肝组织作为正常对照组。通过免疫组织化学（immunohistochemistry,IHC）法检测各组样本AFB1-DNA加合物的表达,并以PCR结合直接测序的方法检测其p53第249密码子的突变情况。结果小肝癌组的AFB1-DNA加合物阳性率最高（73.8%）,显著高于大肝癌组（P=0.016）。而小肝癌组p53第249密码子的突变率（35.7%）却显著低于大肝癌组（P=0.007）。正常对照组AFB1-DNA加合物阳性率为50.0%,但未发现p53第249密码子存在突变。Ⅰ组与Ⅱ组之间无论是AFB1-DNA加合物阳性率还是p53第249密码子的突变率差异均无统计学意义（P1=0.676,P2=1.000）。实验组中,33.3%的样本为AFB1-DNA加合物和p53第249密码子突变双阳性,22.2%的样本为AFB1-DNA加合物和第249密码子突变双阴性。结论广西为AFB1高污染地区,正常人群普遍存在AFB1的暴露。AFB1的暴露可增加HCC的发病概率。p53第249密码子突变可能是影响AFB1相关HCC发生、发展的因素。结合AFB1-DNA加合物表达和第249密码子突变的情况,可有效地了解肝癌患者长期持续性或非持续性的AFB1暴露下DNA的累积损伤情况。     

Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors.

The study was undertaken to determine whether aflatoxin B1 (AFB1)-induced liver tumors in rats produced alpha1-fetoprotein (AFP) and whether the age of the animals would influence such as appearance, a finding suggested by data seen in man. Other liver carcinogens (N-hydroxy-N-2-fluorenylacetamide, N-2-fluorenylacetamide, and diethylnitrosamine) were tested for their ability to induce liver tumors producing AFP. The presence of AFP. The presence of AFP in the serum was determined by double diffusion in agarose and by comparison also by quantitative radioimmunoassay. Using double diffusion, AFP was detected in the majority of tumor-bearing rats that had received either N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide. Sera of diethylinitrosamine-treated rats with liver tumors were all positive, whereas sera of rats bearing AFB1-induced tumors were positive in only a few cases. However, all sera of tumor-bearing rats examined had elevated AFP levels by radioimmunoassay. Nonetheless, the average level of AFP in the sera of rats bearing AFB1-induced tumors was considerably lower, compared to the sera of rats with tumors caused by diethylnitrosamine, N-2-fluorenylacetamide, or N-hydroxy-N2-fluorenylacetamide. Rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, the histological grade of differentiation of inducted tumors did not seem to influence the AFP level.     

Inhibition of ciprofibrate-induced hepatocarcinogenesis in the rat by dimethylthiourea, a scavenger of hydroxyl radical.

DNA damage caused by oxidative stress is considered to play an important role in peroxisome proliferator-induced hepatocarcinogenesis in rats and mice. In this study, we investigated the effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, on ciprofibrate-induced hepatocarcinogenesis. Male F-344 rats were fed a diet containing 0.025% ciprofibrate and given daily intraperitoneal injections of DMTU (5 days a week) at a dose of 50 or 250 mg/kg body weight for 60 weeks at which time the study was terminated. Livers from all animals were analyzed grossly and microscopically for incidence, number and type of tumors. All rats given ciprofibrate alone developed altered areas, neoplastic nodules (NN) and hepatocellular carcinomas (HCC). Combined administration of ciprofibrate and DMTU resulted in inhibition of tumor development. In the group given higher doses of DMTU the incidence of NN was 100% and HCC 0%. The number of tumors per liver also significantly decreased (p<0.001). At lower dose levels DMTU caused significant reduction in the number of tumors per liver (p<0. 05) and a slight reduction (29%) in the incidence of HCC. The inhibitory effect of DMTU on ciprofibrate-induced hepatocarcinogenesis could be explained by hydroxyl radical scavenging properties of DMTU, resulting in decreased free radical induced DNA damage.     

Inhibition of aflatoxin B1-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic preneoplastic foci and tumors by low protein diets: evidence that altered GGT+ foci indicate neoplastic potential.

Previous studies in this laboratory with young Fischer 344 male rats have shown that the post-initiation development of aflatoxin B1 (AFB1)-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic foci was markedly inhibited by low protein feeding, even though the energy intake was greater. This dietary effect, however, did not necessarily apply to hepatic tumor development. Thus, the present investigation was undertaken to examine this dietary effect upon the development of hepatic tumors and, is so doing, to determine the correlation of foci development with tumor development. Following AFB1 dosing (15 daily doses of 0.3 mg/kg each), animals were fed diets containing 6, 14 or 22% casein (5.2, 12.2, 19.1% protein) for 6, 12, 40, 58 and 100 weeks. Foci at 12 weeks and tumors at 40, 58 and 100 weeks developed dose-dependently to protein intake. Foci development, tumor incidence, tumor size and the number of tumors per animal were markedly reduced while the time to tumor emergence was increased with low protein feeding. Non-hepatic tumor incidence also was lower in the animals fed the lowest protein diet. Foci development indices (foci number, per cent liver volume occupied) were highly correlated with tumor incidence at 58 and 100 weeks (r = 0.90-1.00). Tumor and foci inhibition occurred in spite of the greater energy intake.