Aflatoxin and p53 abnormality in duck hepatocellular carcinoma

Recent studies have revealed that a point mutation at codon 249 in the p53 gene predominates in hepatocellular carcinoma (HCC) cases from Southern Africa and China, where infection with hepatitis B virus (HBV) and contamination of aflatoxin B₁ in food are risk factors for HCC. This unique mutation from G to T at the third base in codon 249 observed in human HCC cases is suggested to be linked to aflatoxin exposure. Six ducks with HCC, five of which were fed a diet containing aflatoxin B₁ for 1–2 years, were analysed for the presence of point mutations at this codon of the p53 gene by polymerase chain reaction and direct nucleotide sequencing. None of the six ducks with HCC showed the change at this codon regardless of duck hepatitis B virus infection. This suggests that aflatoxin B₁ itself might not be involved in the unique mutation at codon 249 in hepatocar-cinogenesis, or that other factors coincident with aflatoxin may be responsible for this unique mutation.     

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non HCC prevalent area in China

AIM：In hepatocellular carcinoma(HCC)prevalent areas of China,the point mutation of p53 exon7 is highly correlated with hepatitis B virus(HBV)infection and aflatoxin B intake While in non-HCC_prevalent areas of China,these factors are not so important in the etiology of HCC.Therefore,the point mutation of p53 exon7 may also be different than that in HCC-pervalent areas of China.The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non-HCC-prevalent area in China.METHODS:RCR,PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui,which were confimed by DNA sequencing and Genbak comparison.RESULTS:In the 38 samples of hepatocellular carcinoma,no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases,which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38).The third base mutation(G→T)of p53 codon 249 was found by DNA sequencing and Genbank comparison.CONCLUSION:The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC p53 codon 249 is a hotspot of p53 exon7 point mutation suggesting that the point mutation of p53 exon 7 may not play a majuor role in the carcinogenesis of HCC in Anhui Province,a non-HCC-prevalent area in China.     

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non HCC prevalent area in China

AIM: In hepatocellular carcinoma (HCC) prevalent areas ofChina, the point mutation of p53 exon7 is highly correlatedwith Hepatitis B virus(HBV) infection and aflatoxin B intake.While in non-HCC-prevalent areas of China, these factorsare not so important in the etiology of HCC. Therefore, thepoint mutation of p.53 exon7 may also be different than thatin HCC-prevalent areas of China. The aim of this study is toinvestigate the status and carcinogenic role of the pointmutation of p53 gene exon7 in hepatocellular carcinoma fromAnhui Province, a non-HCC-prevalent area in China.METHODS: PCR,PCR-SSCP and PCR-RFLP were applied toanalyze the homozygous deletion and point mutation of p53exon7 in HCC samples from Anhui, which were confirmedby DNA sequencing and Genbank comparison.RESULTS: In the 38 samples of hepatocellular carcinoma, nohomozygous deletion of p53 exon7 was detected and pointmutations of p53 exon7 were found in 4 cases, which werefound to be heterozygous mutation of codon 249 With amutation rate of 10.53 %(4/38). The third base mutstion(G→T) of p53 codon 249 was found by DNA sequencing andGenbank comparison.CONCLUSION: The incidence of point mutation of p53 codon249 is lower in hepatocellular carcinoma and theheterozygous mutation of p53 exon7 found in these patientsonly indicate that they have genetic susceptibility to HCC.p53 codon 249 is a hotspot of p53 exon7 point mutation,suggesting that the point mutation of p53 exon 7 may notplay a major role in the carcinogenesis of HCC in AnhuiProvince, a non-HCC-prsvalent area in China.     

The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells

A particular point mutation of the tumor suppressor gene p53, namely a G-->T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a human p53 complementary DNA mutated at the third base of codon 249 and analyzed the consequences of this gene transfer on the growth properties of this cell line. HACL-1 cells transfected with mutant p53 showed no increase in their life-span (when compared with HACL-1 cells transfected with the antibiotic resistance gene alone) and did not grow in soft agar, whereas transfection of wild-type p53 into HACL-1 cells led to a proliferation stop. Thus, these results strongly support the view that the mutation at codon 249 of the p53 gene may serve as a fingerprint for aflatoxin B1-induced hepatocellular carcinomas, but is not, by itself, sufficient to immortalize human liver cells.     

Codon 249 mutation in exon 7 of p53 gene in plasma DNA： maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area, China

AIM: One of the characteristics of hepatocellular cardnoma(HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1,20),and it has been identified as a “hotspot” mutation in heptocellular carcinomas occurring in populations exposed to afiatoxin and with high prevalence of hepatitis B virus carriers (2, 3, 9, 10, 16, 24). We evaluated in this paper whether this “hotspot” mutation could be detected in cellfree DNA drculating in plasma of patients with hepatocellular carcinoma and drrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.METHODS: We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200μl of plasma from each sample. The 249^Ser p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.RESULTS: We found in exon 7 of p53 gene G---T transversion at the third base of codon 249 resulting 249^Arg→249^Ser mutation in 10/25 (40%) hepatocellular carcinoma cases,4/20 (20%) cirrhotics, and 2/30 (7%) healthy controls.The adjusted odds ratio for having the mutation was 22.1(95% CI, 3.2-91.7) for HCC cases compared to controls.CONCLUSION: These data show that the 249^Ser p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency,in plasma DNA of Qidong cirrhotics and healthy controls;We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249^Ser p53 mutation should be developed to a new early diagnostic marker for HCC.     

Aflatoxin sufferer and p53 gene mutation in hepatocellular carcinoma

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Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas from Britain.

Human hepatocellular carcinomas from patients in Britain, an area of low prevalence of hepatocellular carcinoma and low dietary exposure to aflatoxin B1, were analyzed for mutations in the p53 tumor-suppressor gene. Abnormalities in the p53 gene were detected in 2 of 19 hepatocellular carcinomas by polymerase chain reaction--single-stranded conformation polymorphism. Direct sequencing of the evolutionarily conserved regions of p53 (exons 5, 6, 7 and 8), where mutations have been commonly found in a variety of tumors, confirmed that only two hepatocellular carcinomas had mutations in p53, one a 6-bp deletion of codons 158 and 159 (exon 5) and the other a G to A transition at codon 286 (exon 8). No mutations were found in any hepatocellular carcinoma in exons 6 and 7; in particular all tumors had wild-type sequence at codon 249, which has been reported to be a mutational hot spot in the p53 gene in hepatocellular carcinomas from high incidence areas such as China and southern Africa. Abnormalities in p53 expression were examined by immunohistochemistry and found in 1 of the 19 hepatocellular carcinomas. These findings show that p53 mutations are infrequently involved in the malignant transformation of hepatocytes in an area of low hepatocellular carcinoma prevalence. They support the suggestion of a possible link between dietary exposure to aflatoxin and selective G to T mutations at codon 249 of the p53 gene. Our observations also indicate that hepatitis B virus infection alone, present in six of the hepatocellular carcinomas examined, does not account for the specificity for codon 249 mutations reported from endemic areas.     

Detection of mutant p53 in hepatocellular cancer from Turkey and its correlation with clinicopathologic parameters

The samples of hepatocellular carcinoma from Turkey, a country with a high prevalence of hepatitis B virus and hepatitis C virus, but low dietary exposure to aflatoxin B1, were examined in order to detect the frequency of mutant p53 and its association with clinical and pathological data. Fifty-two samples of hepatocellular cancer from the patients who were diagnosed in our clinic were included in this study. The mutant p53 protein was searched for by specific enzyme-linked immunosorbent assay. Of 52 patients with hepatocellular carcinoma, 26 (50%) had the mutant p53. The incidence of p53 mutation in hepatocellular cancer patients with chronic liver disease due to hepatitis B virus infection was significantly higher than in those with chronic liver disease due to alcohol, indicating that not alcohol but hepatitis B virus, in fact induces the mutations in p53 gene. In addition, it has been shown that the p53 mutation was significantly associated with the diameter of tumor nodule and the degree of cellular differentiation in hepatocellular cancer. The p53 mutation rate found in our study is concordant for a geography where hepatitis B virus and hepatitis C virus are common. Hepatitis B virus and possibly hepatitis C virus, but not alcohol, should be responsible, to a degree, for the mutational change in p53 protein in hepatocellular cancer patients with chronic liver disease. The p53 mutation is a late event in hepatocarcinogenesis because it is related with cellular differentiation and tumor diameter. The specific ELISA can be a useful screening test in future studies to select the patients for gene therapy using wild-type p53.     

Detection of p53 codon 249 mutation in Nigerian patients with hepatocellular carcinoma using a novel evaluation of cell-free DNA: Original Article

Objectives: This case-control study was done to determine the association and prevalence of p53 codon 249 mutation using cell-free DNA in the plasma of patients with hepatocellular carcinoma (HCC) in South-Western Nigeria. Method: Eighty-five adults with HCC and seventy-seven age and gender matched controls without evidence of liver disease or malignancy involving any part of the body, were recruited. Plasma DNA was analyzed for p53 codon 249 by restriction fragment length polymorphism. Patient evaluation was done by means questionnaire interview, clinical examination, laboratory and radiological tests. The prevalence of the p53 codon 249 mutation was expressed as a percentage amplifiable DNA samples analyzed from HCC patients while that of controls was expressed in the same way. Fisher’s exact test or the student t-test where appropriate were used to assess statistical significance of prevalence between both groups as well as comparison of some characteristics in the HCC cases between those who had codon 249 mutation and those who did not. Associations between the various parameters assessed were determined by odds ratio and significant difference was specified at p < 0.05. Results: p53 codon 249 mutation was present in 6 (7.6%) of the 79 samples from the HCC patients with amplifiable plasma DNA while none (i.e. 0%) of the 73 samples with amplifiable plasma DNA from the controls had this mutation. This prevalence is significantly higher among HCC patients than controls (0.029). The mutation was also found to be significantly associated with HCC (odds ratio = 2.00; 95% C I: 1.70 – 2.35). Conclusion: The prevalence of the p53 codon 249 mutation from plasma DNA of hepatocellular carcinoma patients is significantly higher than among controls in South-Western Nigeria and the presence of this mutation is significantly associated with HCC in this region.     

249ser p53 mutation in the serum of black southern African patients with hepatocellular carcinoma

BACKGROUND AND AIMS: A specific mutation at codon 249 of the p53 tumor suppressor gene (guanine to thymine; arginine to serine [249(serine)p53]) is present in the cell-free plasma of 30-47% of patients with hepatocellular carcinoma (HCC) in regions with uniformly high levels of dietary exposure to the fungal toxin, aflatoxin B(1). No information is available from other regions. We therefore examined cell-free serum from HCC patients in southern Africa, where aflatoxin B(1) exposure ranges from very high to low levels. METHODS: DNA extracted from the serum of 158 black African patients with HCC was amplified by the polymerase chain reaction assay using primers specific for exon 7 of the p53 gene, and submitted to endonuclease cleavage with HaeIII to identify the 249(serine)p53 mutation. The presence of the mutation was confirmed by nucleotide sequencing. RESULTS: The specific mutation was detected in 18% of the patients, giving an odds ratio for HCC in those with the mutation of 13.3 (95% confidence limits 1.8; 100.2). Surprisingly, the mutation was present equally often in rural and urban patients, despite presumed levels of aflatoxin B(1) exposure in the latter being much lower. No correlation was found with the presence of hepatitis B virus infection or the age, sex or tribe of the patients. CONCLUSIONS: The 249(serine)p53 mutation is found less often in the serum of patients with HCC in a region with variable levels of exposure to aflatoxin B(1) than in those with uniformly high levels of exposure, but the mutation does occur in black Africans with presumed lower levels of exposure to the fungal toxin.     

肝细胞癌p53基因249密码子热点突变的研究

目的 研究肝细胞癌(HCC)中p53基因249密码子(p53 E7 cd249)点突变情况。方法 用PCR法及HAEⅢ限制性片段长度多态性分析(HAEⅢ/RFLP)检测河南豫东地区38例HCC石蜡包埋组织及2例肝细胞癌株中p53 E7cd249点突变情况,DNA测序证实。选取广西桂西南地区的10例HCC作对照。结果 来自河南豫东地区的HCC p53 E7 cd249点突变率为10.5％(4/38),对照组广西桂西南地区的HCC p53 E7 cd249点突变为40％(4/10),二者相比具有显著性差异(P<0.05)。2例肝细胞癌株中均未发现HCC p53 E7 cd249点突变。结论 河南豫东地区HCC中p53基因E7 cd249点突变为非高发事件;p53 E7 cd249点突变可能发生在肝细胞癌变的晚期。     

Hepatic aflatoxin B1‐DNA adducts and TP53 mutations in patients with hepatocellular carcinoma despite low exposure to aflatoxin B1 in southern Japan

Background & aims: Hepatitis B or C virus infection is considered to be the main cause of hepatocellular carcinoma (HCC) in Japan. Aflatoxin B1 (AFB1) is a carcinogen associated with HCC in regions with high exposure. Mutations in codon 249, exon 7 are a hallmark of AFB1 exposure. Therefore, to clarify the role of AFB1 in hepatocarcinogenesis, we examined AFB1‐DNA in liver tissue and sequenced TP53 in Japanese patients with HCC. Methods: Hepatocyte AFB1‐DNA adducts were determined immunohistochemically and direct sequencing of TP53 was done to determine mutations in 188 of 279 patients who underwent hepatic resection for HCC. We assessed hepatitis C virus antibodies (HCV Ab) and HBSAg expression; patients without either were defined as having non‐B non‐C hepatocellular carcinoma (NBNC HCC). Results: AFB1‐DNA adducts were detected in hepatocyte nuclei in 18/279 patients (6%), including13/83 patients (16%) with NBNC HCC and 5/51 patients (10%) expressing hepatitis B surface antigen. None of the patients with HCV Ab (n=136) were positive for AFB1‐DNA. The incidence of the G–T transversion and mutations in exon 7 of TP53 in patients with AFB1‐DNA adducts were significantly higher in patients with than in patients without AFB1‐DNA adducts. All three patients with the codon 249 AGG–AGT mutation had AFB1‐DNA adducts. Conclusion: Although exposure to AFB1 is thought to be low in Japan, it is still associated with hepatocarcinogenesis, particularly in NBNC HCC and hepatitis B individuals.     

Geographic characterization of hepatitis virus infections,genotyping of hepatitis B virus,and p53 mutation in hepatocellular carcinoma analyzed by in situ detection of viral genomes from carcinoma tissues: comparison among six different countries

We investigated the relationship of infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) to p53 mutation in hepatocellular carcinomas (HCC) from six countries, including Japan, China, Korea, Vietnam, Spain, and the Unites States. For this purpose, we used formalin-fixed, paraffin-embedded liver tissues obtained from 449 patients with HCC to detect the viral and p53 genes by polymerase chain reaction (PCR). HBV was the most prevalent in Korea (69.1%), China (66.1%), Vietnam (60.5%), and Spain (38.6%). In contrast, HCV was the most prevalent in Japan (59.8%) and in the United States (41.5%). Type C of HBV was the most common genotype (78.6%) encountered in HCC in these countries. Importantly, among 125 intrahepatic HBV DNA-positive patients, 44 (35.2%) were serologically negative for HBsAg (occult hepatitis B). Based on PCR, immunohistochemical, serological, and clinical findings, 4.8% of HCC patients were diagnosed with non-B, non-C. A point mutation at exon 7 of p53 was detected in 20 of the 239 HCC samples examined, including those from 9 Chinese, 5 American, 2 Japanese, 2 Korean, and 2 Spanish patients, respectively. Interestingly, a point mutation with an amino acid substitution at codon 251 (Ile-->Asn) was detected frequently in 11 of 20 (55%) cases. A specific mutation induced by Aflatoxin B1 at codon 249 was seen in two patients, both Chinese. Our results suggest that genotype C of HBV may play an important role in hepatocarcinogenesis in different geographic regions, and that in situ detection of HBV genomes could be important for clarifying the agent(s) of unknown etiology related to HCC.     

Low frequency of p53 mutations observed in a diverse collection of primary hepatocellular carcinomas.

Recent studies of the p53 tumor suppressor locus (designated TP53) in primary hepatocellular carcinoma (PHC) have identified a high frequency of codon 249 mutations. Due to the geographic location from which the samples were obtained and the substitution observed, the mutation was suggested to be attributable to aflatoxin B1 (AFB1) exposure. To determine the generality of this phenomenon, we have examined PHC tissues from 107 geographically and ethnically diverse sources. The frequency of p53 gene mutations was evaluated by using PCR/restriction-digest methods, GC-clamp (G+C-rich sequence) denaturing gradient gel electrophoresis, and DNA sequencing. The mutation rate observed in tumors from high-AFB1-exposure regions (25%) was more than double the rate observed in low-exposure regions (12%) but lower than the 50% frequency previously reported. Codon 249 mutations occurred at a much lower frequency than previously reported (2 of 107 samples examined). These results suggest that changes in DNA encoding p53 may not represent primary oncogenic effects but instead represent genetic changes related to tumor progression. High AFB1 levels may facilitate the generation of these progressional changes, but not by inducing a specific p53 gene mutation at codon 249 as previously reported.     

Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes.

Approximately half of hepatocellular carcinoma (HCC) from regions in the world with high contamination of food with the mycotoxin aflatoxin B1 (AFB1) contain a mutation in codon 249 of the p53 tumor suppressor gene. The mutation almost exclusively consists of a G-->T transversion in the third position of this codon, resulting in the insertion of serine at position 249 in the mutant protein. To gain insight into the mechanism of formation of this striking mutational hot spot in hepatocarcinogenesis, we studied the mutagenesis of codons 247-250 of p53 by rat liver microsome-activated AFB1 in human HCC cells HepG2 by restriction fragment length polymorphism/polymerase chain reaction genotypic analysis. AFB1 preferentially induced the transversion of G-->T in the third position of codon 249. However, AFB1 also induced G-->T and C-->A transversions into adjacent codons, albeit at lower frequencies. Since the latter mutations are not observed in HCC it follows that both mutability on the DNA level and altered function of the mutant serine 249 p53 protein are responsible for the observed mutational hot spot in p53 in HCC from AFB1-contaminated areas. Our results are in agreement with an etiological role of AFB1 in hepatocarcinogenesis in regions of the world with AFB1-contaminated food.     

人p53/荧光蛋白融合基因在高转移人肝癌细胞中的表达与定位

目的 观察Ｐ５３基因在肝癌细胞中的正常表达及定位情况。方法 根据野生型Ｐ５３基因的编码顺序,ＰＣＲ主增突变的ＷＴ－Ｐ５３基因,使其３＇端的终止密友ＴＧＡ突变为ＴＧＡ突变为ＴＧＧ,用基因重组技术将共与绿色荧光蛋白（ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｃｅ ｐｒｏｔｅｌｎ,ＧＦＰ）融合,通过真核表达质粒－脂质体介导,导入人高转移肝癌细胞系ＭＨＣＣ９７（ＰＣＲ检测有Ｐ５３基因突变）中、用荧光显微镜观察Ｐ５３     

Codon 249 mutations of p53 gene in development of hepatocellular carcinoma

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Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong,China

We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men.     

Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1.

Aflatoxin B1 (AFB1) has been postulated to be a hepatocarcinogen in humans, possibly by causing p53 mutations at codon 249. AFB1 is metabolized via the phase I and II detoxification pathways; hence, genetic variation at those loci may predict susceptibility to the effects of AFB1. To test this hypothesis, genetic variation in two AFB1 detoxification genes, epoxide hydrolase (EPHX) and glutathione S-transferase M1 (GSTM1), was contrasted with the presence of serum AFB1-albumin adducts, the presence of hepatocellular carcinoma (HCC), and with p53 codon 249 mutations. Mutant alleles at both loci were significantly overrepresented in individuals with serum AFB1-albumin adducts in a cross-sectional study. Mutant alleles of EPHX were significantly overrepresented in persons with HCC, also in a case-control study. The relationship of EPHX to HCC varied by hepatitis B surface antigen status and indicated that a synergistic effect may exist. p53 codon 249 mutations were observed only among HCC patients with one or both high-risk genotypes. These results indicate that individuals with mutant genotypes at EPHX and GSTM1 may be at greater risk of developing AFB1 adducts, p53 mutations, and HCC when exposed to AFB1. Hepatitis B carriers with the high-risk genotypes may be an even greater risk than carriers with low-risk genotypes. These findings support the existence of genetic susceptibility in humans to the environmental carcinogen AFB1 and indicate that there is a synergistic increase in risk of HCC with the combination of hepatitis B virus infection and susceptible genotype.