aflatoxin-b1-formamidopyrimidine and Carcinoma--Hepatocellular

Aflatoxin B1 (AFB1) is a known carcinogen associated with early-onset hepatocellular carcinoma (HCC) and is thought to contribute to over half a million new HCCs per year. Although some of the fundamental risk factors are established, the molecular basis of AFB1-induced mutagenesis in primate cells has not been rigorously investigated. To gain insights into genome instability that is produced as a result of replicating DNAs containing AFB1 adducts, site-specific mutagenesis assays were used to establish the mutagenic potential of the persistent ring-opened AFB1 adduct, AFB1-formamidopyrimidine (AFB1-FAPY). This lesion was highly mutagenic, yielding replication error frequencies of 97%, with the predominant base substitution being a G to T transversion. This transversion is consistent with previous mutational data derived from aflatoxin-associated HCCs. In vitro translesion synthesis assays demonstrated that polymerase (pol) ζ was the most likely candidate polymerase that is responsible for the G to T mutations induced by this adduct.

Topics: Aflatoxin B1; Animals; Carcinoma, Hepatocellular; Chlorocebus aethiops; COS Cells; DNA Adducts; DNA Replication; DNA, Single-Stranded; Humans; Liver Neoplasms; Mutagenesis, Site-Directed; Mutation; Polymerase Chain Reaction; Pyrimidines

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B(1) (AFB(1)). Hypotheses have been put forth that AFB(1), in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB(1) is 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxyaflatoxin B(1) (AFB(1)-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB(1)-formamidopyrimidine (AFB(1)-FAPY) adduct. AFB(1)-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB(1) exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB(1)-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB(1)-N7-Gua, and (ii) one proposed rotamer of AFB(1)-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.

Topics: Aflatoxin B1; Bacteriophage M13; Carcinoma, Hepatocellular; DNA Adducts; DNA, Neoplasm; DNA, Viral; Guanine; Humans; Liver Neoplasms; Molecular Structure; Mutagenesis; Mutagens; Pyrimidines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The presence of aflatoxin-B1-formamidopyrimidine, a persistent aflatoxin-deoxyribonucleic acid (DNA) adduct, was investigated in vivo by immunohistochemical analysis in 14 paired hepatocellular carcinoma and nontumorous human liver tissue sections using a monoclonal anti-aflatoxin-B1-formamidopyrimidine antibody. Nuclear and cytoplasmic accumulations of adducts were found in 4 of 14 nontumorous specimens but in none of 14 tumorous tissues and in none of three normal control livers. In vitro adduct formation and cellular DNA was investigated with a modified DNA immunoblot assay. These studies revealed (a) no significant difference in the amount of adduct formed by DNA samples with or without integrated hepatitis B virus DNA, (b) no difference in the amount of adduct formed with DNA from either tumorous or nontumorous tissues from a given individual, and (c) remarkable and reproducible differences in the capacity of DNA from different individuals to form in vitro adducts. Our DNA immunoblot assay will facilitate further studies on the molecular role of aflatoxin-B1-form-amidopyrimidine adducts in human hepatocarcinogenesis.

Topics: Aflatoxin B1; Aflatoxins; Antibodies, Monoclonal; Carcinogens; Carcinoma, Hepatocellular; DNA; DNA, Neoplasm; Humans; Immunoblotting; Liver Neoplasms; Pyrimidines