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AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249

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ABSTRACT

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at –CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

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Cytosine methylation at codon 248 (-CGG-) of p53 gene sensitizes codon 249 (-AGG-) for Acet and Cro modificationsSingle 5’-end A. or 3’-end B. 32P labeled exon 7 p53 DNA fragments were methylated with CpG methylase, as previously described [13, 61] then modified with Acet (100 μM, 1 h at 25 °C) and Cro (100 μM, 1 h at 37°C). PdG distributions in the fragments were mapped by the UvrABC incision method [26]. ∧C and C represent the methylated and unmethylated DNA fragments, respectively. * indicates the UvrABC incised base. C. The effect of methylation on Acet and Cro induced PdG formation. The relative levels of PdG formation at different sequences of 3’-end 32P-labled exon 7 of p53 gene fragments with (Methyl) and without (Un-methyl) CpG methylation as shown in (B) were quantified as described [13, 36]. RI represents relative intensity. Note: CpG methylation greatly enhances Acet- and Cro-induced PdG formation at codon 249.
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Figure 3: Cytosine methylation at codon 248 (-CGG-) of p53 gene sensitizes codon 249 (-AGG-) for Acet and Cro modificationsSingle 5’-end A. or 3’-end B. 32P labeled exon 7 p53 DNA fragments were methylated with CpG methylase, as previously described [13, 61] then modified with Acet (100 μM, 1 h at 25 °C) and Cro (100 μM, 1 h at 37°C). PdG distributions in the fragments were mapped by the UvrABC incision method [26]. ∧C and C represent the methylated and unmethylated DNA fragments, respectively. * indicates the UvrABC incised base. C. The effect of methylation on Acet and Cro induced PdG formation. The relative levels of PdG formation at different sequences of 3’-end 32P-labled exon 7 of p53 gene fragments with (Methyl) and without (Un-methyl) CpG methylation as shown in (B) were quantified as described [13, 36]. RI represents relative intensity. Note: CpG methylation greatly enhances Acet- and Cro-induced PdG formation at codon 249.

Mentions: To determine whether sequence context and/or epigenetic modifications lead to the preferential binding of Acet and Cro at codon 249 in the p53 gene in AFB1, Acet- and Cro- treated HepG2 cells, we mapped the meth-OH-PdG distribution in Acet- and Cro-modified PCR amplified DNA fragments of exon 7 of the p53 gene, with and without cytosine methylation at -CG- sites. It has been established that cytosine methylation at CpG sites affects DNA adduct formation at the CpG sites as well as at the neighboring DNA sequences [13, 37, 38]. The results in Figure 3 show that Acet and Cro induced very little, if any, meth-OH-PdG adducts at codon 249 (-AGG-)in unmethylated DNA fragments. In contrast, both Acet and Cro induced high levels of meth-OH-PdG adducts at codon 249 in 5’-CG- methylated DNA fragments. Methylation at 5’-CG- sites did not affect Acet- and Cro-induced PdG formation at the codon 248 (-CGG-); however, methylation at the 5’-CG-sites does enhance meth-OH-PdG formation at other sequences and the observed effect was particularly dramatic at codon 249. These results indicate that cytosine methylation at the 5’-CG- site of codon 248 sensitizes codon 249 to Acet and Cro modifications to form meth-OH-PdG adducts.


AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249
Cytosine methylation at codon 248 (-CGG-) of p53 gene sensitizes codon 249 (-AGG-) for Acet and Cro modificationsSingle 5’-end A. or 3’-end B. 32P labeled exon 7 p53 DNA fragments were methylated with CpG methylase, as previously described [13, 61] then modified with Acet (100 μM, 1 h at 25 °C) and Cro (100 μM, 1 h at 37°C). PdG distributions in the fragments were mapped by the UvrABC incision method [26]. ∧C and C represent the methylated and unmethylated DNA fragments, respectively. * indicates the UvrABC incised base. C. The effect of methylation on Acet and Cro induced PdG formation. The relative levels of PdG formation at different sequences of 3’-end 32P-labled exon 7 of p53 gene fragments with (Methyl) and without (Un-methyl) CpG methylation as shown in (B) were quantified as described [13, 36]. RI represents relative intensity. Note: CpG methylation greatly enhances Acet- and Cro-induced PdG formation at codon 249.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Cytosine methylation at codon 248 (-CGG-) of p53 gene sensitizes codon 249 (-AGG-) for Acet and Cro modificationsSingle 5’-end A. or 3’-end B. 32P labeled exon 7 p53 DNA fragments were methylated with CpG methylase, as previously described [13, 61] then modified with Acet (100 μM, 1 h at 25 °C) and Cro (100 μM, 1 h at 37°C). PdG distributions in the fragments were mapped by the UvrABC incision method [26]. ∧C and C represent the methylated and unmethylated DNA fragments, respectively. * indicates the UvrABC incised base. C. The effect of methylation on Acet and Cro induced PdG formation. The relative levels of PdG formation at different sequences of 3’-end 32P-labled exon 7 of p53 gene fragments with (Methyl) and without (Un-methyl) CpG methylation as shown in (B) were quantified as described [13, 36]. RI represents relative intensity. Note: CpG methylation greatly enhances Acet- and Cro-induced PdG formation at codon 249.
Mentions: To determine whether sequence context and/or epigenetic modifications lead to the preferential binding of Acet and Cro at codon 249 in the p53 gene in AFB1, Acet- and Cro- treated HepG2 cells, we mapped the meth-OH-PdG distribution in Acet- and Cro-modified PCR amplified DNA fragments of exon 7 of the p53 gene, with and without cytosine methylation at -CG- sites. It has been established that cytosine methylation at CpG sites affects DNA adduct formation at the CpG sites as well as at the neighboring DNA sequences [13, 37, 38]. The results in Figure 3 show that Acet and Cro induced very little, if any, meth-OH-PdG adducts at codon 249 (-AGG-)in unmethylated DNA fragments. In contrast, both Acet and Cro induced high levels of meth-OH-PdG adducts at codon 249 in 5’-CG- methylated DNA fragments. Methylation at 5’-CG- sites did not affect Acet- and Cro-induced PdG formation at the codon 248 (-CGG-); however, methylation at the 5’-CG-sites does enhance meth-OH-PdG formation at other sequences and the observed effect was particularly dramatic at codon 249. These results indicate that cytosine methylation at the 5’-CG- site of codon 248 sensitizes codon 249 to Acet and Cro modifications to form meth-OH-PdG adducts.

View Article: PubMed Central - PubMed

ABSTRACT

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at –CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

No MeSH data available.


Related in: MedlinePlus