Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems.
Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.
Denissenko
M F
MF
Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Cahill
J
J
Koudriakova
T B
TB
Gerber
N
N
Pfeifer
G P
GP
D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000032:analysis* / Q000633:toxicity; D000818:Animals; D001711:Biotransformation; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D016158:Genes, p53 / Q000187:drug effects; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D008099:Liver / Q000737:chemistry / Q000187:drug effects; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley
D018736:DNA Adducts; C072611:aflatoxin B1-DNA adduct; C006426:aflatoxin B1-2,3-oxide; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A