Preferential Promutagenic Lesions at Exons 7-8 of Human P53 Genomic DNA Induced by the Direct-Acting Hepatocarcinogens N-Nitroso-2-Acetylaminofluorene

Abstract

Reaction of isolated plasmid (php53B) DNA containing human p53 gene with NO-AAF or AcO-AAF produced the corresponding DNA adducts. DNA polymerase fingerprint analysis (DPFA) was employed for identifying the substituted bases in the DNA-carcinogen adducts. The following results were obtained: two “hot regions” at codons 223-250 or 257-283 of p53 gene were more easily attacked by NO-AAF or AcO-AAF. The mutation hot spots in various human hepatocellular carcinomas including 175, 248, 249 and 280 codons were also strongly attacked by NO-AAF or AcO-AAF. The enother plasmid DNA (pUC 19) containing lac gene was also reacted with NO-AAF or AcO-AAF to yield pemutational lesions. After transfection into E. coli JM109 cells, the induced lac gene mutants were selected with X-Gal plate as indicated by the appearance of white colonies. Unlike human p53 gene, the premutational lesions in lac gene induced by NO-AAF or AcO-AAF targeting were rather randomly distributed. The DNA sequences of 33 mutants in NO-AAF group and 29 mutants in AcO-AAF group were analyzed and no mutational hot regions were found in the lac gene. These findings indicated that human p53 gene has 2 regions through exons 7-8 were more easily attacked by hepatocarcinogens. Such results were correlated with the clinical findings that high p53 gene mutations in exons 7-8 were occurred in human hepatocellular carcinoma.