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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.

No MeSH data available.


Repair kinetics of AFB1-induced DNA damage in the overall genomic DNA and in the coding strand of p53 gene exon 7 in human hepatocytesHepG2 cells were treated with AFB1 (100 μM, 1 h at 37 °C), then incubated for different time periods (0, 2, 4, 8, and 16 h). A. PdG and AFB1-E-dG adducts formed in the genomic DNA were detected by antibody and quantum dot labeling method [20, 33, 34]. AFB1-E or PdG standards (Std) are included for comparison. Amount of input DNA stained by methylene blue. B. Repair kinetics of PdG adducts and AFB1-E-dG adducts in the overall genomic DNA. C. Mapping DNA damage distribution at sequence level in the coding strand of p53 exon 7 by the UvrABC/LMPCR after different repair times, as described in Figure 2 [36]. D. Repair kinetics of DNA damage formed in codons 240, 243, 244, 245, 248, and 249. Note: AFB1-E-dG repair is faster than meth-OH-PdG repair. Repair of meth-OH-PdG in codon 249 is poorer than in other codons.
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Figure 4: Repair kinetics of AFB1-induced DNA damage in the overall genomic DNA and in the coding strand of p53 gene exon 7 in human hepatocytesHepG2 cells were treated with AFB1 (100 μM, 1 h at 37 °C), then incubated for different time periods (0, 2, 4, 8, and 16 h). A. PdG and AFB1-E-dG adducts formed in the genomic DNA were detected by antibody and quantum dot labeling method [20, 33, 34]. AFB1-E or PdG standards (Std) are included for comparison. Amount of input DNA stained by methylene blue. B. Repair kinetics of PdG adducts and AFB1-E-dG adducts in the overall genomic DNA. C. Mapping DNA damage distribution at sequence level in the coding strand of p53 exon 7 by the UvrABC/LMPCR after different repair times, as described in Figure 2 [36]. D. Repair kinetics of DNA damage formed in codons 240, 243, 244, 245, 248, and 249. Note: AFB1-E-dG repair is faster than meth-OH-PdG repair. Repair of meth-OH-PdG in codon 249 is poorer than in other codons.

Mentions: We then used antibodies that are specific for PdG and AFB1-E-dG to determine the repair kinetics of these two types of DNA adducts induced by AFB1 in the hepatocytes. The results in Figure 4A & 4B show that the repair kinetics of AFB1-E-dG adducts are faster than for meth-OH-PdG adducts in the AFB1-treated HepG2 cells. Using the UvrABC incision method in combination with LMPCR we then determined the repair kinetics of AFB1-induced DNA damage at the coding strand (non-transcribed strand) of the p53 gene exon 7 in hepatocytes. Figure 4C & 4D show that the repair of DNA damage induced by AFB1 at codon 249 is much slower and less efficient than the repair of adducts formed in the genomic DNA overall and specifically in codons 240, 243, 244, 245, and 248. The repair kinetics of DNA damage formed at codons containing a –CpG- sequence (codons 243, 244, 245, and 248) is slower than at a codon without a -CpG- sequence (codon 240). These results are consistent with our previous finding that the repair of bulky DNA damage formed at –CpG- sites is slower than repair of bulky DNA damage formed at the non-CpG sites [39].


AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249
Repair kinetics of AFB1-induced DNA damage in the overall genomic DNA and in the coding strand of p53 gene exon 7 in human hepatocytesHepG2 cells were treated with AFB1 (100 μM, 1 h at 37 °C), then incubated for different time periods (0, 2, 4, 8, and 16 h). A. PdG and AFB1-E-dG adducts formed in the genomic DNA were detected by antibody and quantum dot labeling method [20, 33, 34]. AFB1-E or PdG standards (Std) are included for comparison. Amount of input DNA stained by methylene blue. B. Repair kinetics of PdG adducts and AFB1-E-dG adducts in the overall genomic DNA. C. Mapping DNA damage distribution at sequence level in the coding strand of p53 exon 7 by the UvrABC/LMPCR after different repair times, as described in Figure 2 [36]. D. Repair kinetics of DNA damage formed in codons 240, 243, 244, 245, 248, and 249. Note: AFB1-E-dG repair is faster than meth-OH-PdG repair. Repair of meth-OH-PdG in codon 249 is poorer than in other codons.
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Figure 4: Repair kinetics of AFB1-induced DNA damage in the overall genomic DNA and in the coding strand of p53 gene exon 7 in human hepatocytesHepG2 cells were treated with AFB1 (100 μM, 1 h at 37 °C), then incubated for different time periods (0, 2, 4, 8, and 16 h). A. PdG and AFB1-E-dG adducts formed in the genomic DNA were detected by antibody and quantum dot labeling method [20, 33, 34]. AFB1-E or PdG standards (Std) are included for comparison. Amount of input DNA stained by methylene blue. B. Repair kinetics of PdG adducts and AFB1-E-dG adducts in the overall genomic DNA. C. Mapping DNA damage distribution at sequence level in the coding strand of p53 exon 7 by the UvrABC/LMPCR after different repair times, as described in Figure 2 [36]. D. Repair kinetics of DNA damage formed in codons 240, 243, 244, 245, 248, and 249. Note: AFB1-E-dG repair is faster than meth-OH-PdG repair. Repair of meth-OH-PdG in codon 249 is poorer than in other codons.
Mentions: We then used antibodies that are specific for PdG and AFB1-E-dG to determine the repair kinetics of these two types of DNA adducts induced by AFB1 in the hepatocytes. The results in Figure 4A & 4B show that the repair kinetics of AFB1-E-dG adducts are faster than for meth-OH-PdG adducts in the AFB1-treated HepG2 cells. Using the UvrABC incision method in combination with LMPCR we then determined the repair kinetics of AFB1-induced DNA damage at the coding strand (non-transcribed strand) of the p53 gene exon 7 in hepatocytes. Figure 4C & 4D show that the repair of DNA damage induced by AFB1 at codon 249 is much slower and less efficient than the repair of adducts formed in the genomic DNA overall and specifically in codons 240, 243, 244, 245, and 248. The repair kinetics of DNA damage formed at codons containing a –CpG- sequence (codons 243, 244, 245, and 248) is slower than at a codon without a -CpG- sequence (codon 240). These results are consistent with our previous finding that the repair of bulky DNA damage formed at –CpG- sites is slower than repair of bulky DNA damage formed at the non-CpG sites [39].

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.