aflatoxin-b1-formamidopyrimidine and Liver-Neoplasms

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

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.

Topics: Aflatoxin B1; Animals; Carcinogens; Cocarcinogenesis; DNA; DNA Repair; Female; Genetic Predisposition to Disease; Guanine; Liver; Liver Neoplasms; Lung; Lung Neoplasms; Male; Mice; Pyrimidines; Rats; Rats, Sprague-Dawley; Species Specificity

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

Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.

Topics: Adolescent; Adult; Aflatoxin B1; Aged; Aged, 80 and over; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Chromatography, High Pressure Liquid; Cytosol; Dinitrochlorobenzene; DNA; Female; Glutathione Transferase; Humans; Liver; Liver Neoplasms; Lymphocytes; Male; Middle Aged; Polymorphism, Genetic; Pyrimidines; Stilbenes

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