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, 99 (10), 6655-60

The Aflatoxin B(1) Formamidopyrimidine Adduct Plays a Major Role in Causing the Types of Mutations Observed in Human Hepatocellular Carcinoma

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The Aflatoxin B(1) Formamidopyrimidine Adduct Plays a Major Role in Causing the Types of Mutations Observed in Human Hepatocellular Carcinoma

Maryann E Smela et al. Proc Natl Acad Sci U S A.

Abstract

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.

Figures

Figure 1
Figure 1
Aflatoxin biochemistry. AFB1 is activated to the reactive exo-8,9-epoxide form by cytochrome P450 in the liver. The epoxide can react at the N7 position of guanine in DNA to form the primary AFB1-N7-Gua adduct. This product can break down to form two secondary lesions, the AP site and the ring-opened AFB1-FAPY adduct. The FAPY adduct itself consists of two proposed rotameric forms, FAPY major and FAPY minor. FAPY minor is formed first, subsequently equilibrating to a 2:1 ratio of FAPY major:FAPY minor. The FAPY major structure can be found in Stone and coworkers (29), and the FAPY minor structure is yet unresolved, but one possible structure is depicted here. (Inset) Separation of AFB1-FAPY rotamers. HPLC was performed on a 13-base oligonucleotide containing one single AFB1-FAPY adduct. Under the HPLC conditions described, FAPY major can be separated from FAPY minor. The equilibrium mixture shown here is roughly 2:1 major to minor. This mixture was maintained throughout the experiments described in this article and is referred to as FAPY mix. The major and minor peaks were collected and tested as indicated.
Figure 2
Figure 2
Lethality and mutagenicity of AFB1-FAPY. (A) M13 genomes contained a single aflatoxin adduct and were replicated in E. coli expressing different bypass polymerases. (B) Cell strains challenged with FAPY mix, FAPY major, or an unmodified control exhibited different levels of survival. The x axis depicts the adduct with which the cell was challenged and the polymerase status of the cell, whereas the y axis is the number of infective centers per milliliter. (C) The G to T mutation frequency for cell strains challenged with FAPY mix is represented here. Cell strains expressed the bypass polymerases indicated by the x axis. The G to T mutation frequency was calculated by counting the number of light blue plaques and dividing by the total number of blue plaques observed. Error bars for both graphs reflect the SEM for four sets of data.

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