Ligation-Mediated Polymerase Chain Reaction Detection of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine and 5-Hydroxycytosine at the Codon 176 of the p53 Gene of Hepatitis C-Associated Hepatocellular Carcinoma Patients.

Andrea Galli,Armelle Munnia,Roger Godschalk,Elisabetta Ceni,Filippo Cellai,Frederik Jan Van Schooten,Roger W Giese,Marco Peluso,Mirko Tarocchi

doi:10.3390/ijms21186753

Abstract

Molecular mechanisms underlying Hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) pathogenesis are still unclear. Therefore, we analyzed the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and other oxidative lesions at codon 176 of the p53 gene, as well as the generation of 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG), in a cohort of HCV-related HCC patients from Italy. Detection of 8-oxodG and 5-hydroxycytosine (5-OHC) was performed by ligation mediated-polymerase chain reaction assay, whereas the levels of M1dG were measured by chromatography and mass-spectrometry. Results indicated a significant 130% excess of 8-oxodG at –TGC– position of p53 codon 176 in HCV-HCC cases as compared to controls, after correction for age and gender, whereas a not significant increment of 5-OHC at –TGC– position was found. Then, regression models showed an 87% significant excess of M1dG in HCV-HCC cases relative to controls. Our study provides evidence that increased adduct binding does not occur randomly on the sequence of the p53 gene but at specific sequence context in HCV-HCC patients. By-products of lipid peroxidation could also yield a role in HCV-HCC development. Results emphasize the importance of active oxygen species in inducing nucleotide lesions at a p53 mutational hotspot in HCV-HCC patients living in geographical areas without dietary exposure to aflatoxin B1.

Highlights

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the second leading cause of cancer death [1]
The formation of M1dG in the treated calf-thymus DNA sample was confirmed by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [21]
HCC cases relatiinveHtCoCcpoantiternotslsa,nadftceorntarodlsju, sretsipnegctifvoerlya. gMeulatinvadrigateenadnaelry.seWs uhseinng slotag-tnisotrimcaall raengraeslysiosnes were repeated after excluding tmchoonedtr3eolslosh,baaefvtesereaalssdoujusbshtjoienwcgntfsothrwaatgiaetnhan8s7dt%egeaenxtdcoeessris.sWo,fhaMedn1disGstaetoiacssctiuecraroleafdnteianlnyHsceCshVwa-rereraleactrteeedpreHiazCteeCddcaabfstyeesrhreexilgcalthuivdReintOog S and lipid peroxidation (LPO) [23], no significant chthaen3goebseswe esurbejefcotsuwnitdh isnteatthoseisr, easduisletsas.e often characterized by high reactive oxygen species (ROS) and LPO [23], no significant changes were found in the results

Summary

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the second leading cause of cancer death [1]. In Asia and Africa, hepatitis B virus infection and dietary exposure to AFB1 has been associated with G:C to T:A transversions at the third base in codon 249 of the p53 gene causing R249S substitution. Outside the R249S mutation, G:C to T:A transversions at codon 176 can be over-represented in 28–39% of HCV-related HCCs [2]. In this case, a direct mutagenic effect of HCV proteins, that deregulate host cell cycle checkpoints and induce virus and immune-mediated oxidative stress [7], leading to somatic mutations in hepatic cells has been suggested [8]. Significant increments of 8-oxodG at the mutational hotspot codons 163 and 175 were found in breast cancer women relative to controls, indicating a causal relationship between adducts and breast cancer

Methods

Results

Conclusion