An Investigation of Point Mutations at 7th Exon of Gene P53 in Hepatocellular Carcinoma Patients in Kermanshah Province and the Study of Mutation in Liver Specimens of Mice Exposed to Aflatoxin B1

摘要

Hepatocellular Carcinoma (HCC) is the fifth most common malignancy in the world and the third most common cause of cancer related death worldwide. Incidence of HCC is different in various parts of the world depending on gender, location, dietary aflatoxin exposure and chronic hepatitis B and C infections. The point mutation in p53 gene, 7 exon, 249 codon (AGG AGT, which leads to the substitution of the remaining argenine to serine) has the highest frequency in patients affected with HCC. Among factors associated with HCC, AFB1 is considered to be one of the most important causative agents in the formation of HCC in regions with HBV infection and AFB1 exposed dietary. The aim of this study, is to investigate the status and carcinogenic role of p53 gene, 7 exon and point mutation in HCC affected patients in Kermanshah and the AFB1 treated mice. Twenty five Formalin-Fixed Paraffin-Embedded (FFPT) tissues related to HCC patients were collected from pathology centers, which were diagnosed using histological methods. Moreover, 16 liver specimens of mice exposed to AF were gathered. Extracted DNA from these cases was amplified by PCR with specific primers. Furthermore to analyze the point mutation of p53 exon 7 in human HCC samples and mices liver specimens, PCR-RFLP and PCR-SSCP were applied. According to the RFLP results, for human samples that were cleaved by restriction enzyme, HaeIII (which cuts GG CC within codon 249 of exon 7) no point mutations were found and all samples were cleaved by this enzyme. The PCR-SSCP results showed two mutations in other codons of exon 7. Also, SSCP for mice PCR products showed that there are clear differences between control specimen and AFB1 treated samples. This indicated that in addition to the previously mentioned nucleotide mutation, mutation has also occurred in other nucleotides of exon 7. The results of this study indicate that there are not enough evidences to prove the correlation between AFB1 and HCC index in Kermanshah. However, finding mutation in other codons may suggest the contribution of other risk factors rather than exposure to aflatoxin B1 to the incidence of HCC. By performing statistical studies, no significant correlation was observed between p53 mutation and histological grade, tumor stage, sex and age. However, there is a direct relationship between these mutations and cirrhosis. PCR results of extracted DNA from the liver of mice exposed to aflatoxin B1 showed that the used dosage of aflatoxin B1 in this study is carcinogenesis.