A single site-specific trans-opened 7,8,9,10-tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct induces mutations at multiple sites in DNA.
Site-specific mutagenicity of trans-opened adducts at the exocyclic N(2)-amino group of guanine by the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-enantiomers of a benzo[a]pyrene 7,8-diol 9,10-epoxide (7-hydroxyl and epoxide oxygen are trans, BPDE-2) has been determined in Chinese hamster V79 cells and their repair-deficient counterpart, V-H1 cells. Four vectors containing single 10S-BPDE-dG or 10R-BPDE-dG adducts positioned at G(0) or G(-1) in the analyzed 5'-ACTG(0)G(-1)GA sequence of the non-transcribed strand were separately transfected into the cells. Mutations at each of the seven nucleotides were analyzed by a novel primer extension assay using a mixture of one dNTP complementary to the mutated nucleotide and three other ddNTPs and were optimized to quantify levels of a mutation as low as 1%. Only G --> T mutations were detected at the adducted sites; the 10S adduct derived from the highly carcinogenic (+)-diol epoxide was 40-50 and 75-140% more mutagenic than the 10R adduct in V79 and V-H1 cells, respectively. Importantly, the 10S adducts, but not the 10R adducts, induced separate non-targeted mutations at sites 5' to the G(-1) and G(0) lesions (G(0) --> T and C --> T, respectively) in both cell lines. Neither the T 5' to G(0) nor sites 3' to the lesions showed mutations. Non-targeted mutations may enhance overall mutagenicity of the 10S-BPDE-dG lesion and contribute to the much higher carcinogenicity and mutagenicity of (+)-BPDE-2 compared with its (-)-enantiomer. Our study reports a definitive demonstration of mutations distal to a site-specific polycyclic aromatic hydrocarbon adduct.
Kramata
Pavel
P
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854-8020, USA. kramata@rci.rutgers.edu
Zajc
Barbara
B
Sayer
Jane M
JM
Jerina
Donald M
DM
Wei
Caroline S-J
CS
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D018736:DNA Adducts / Q000235:genetics*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives*; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D013237:Stereoisomerism; D014162:Transfection
D018736:DNA Adducts; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C040060:7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D003849:Deoxyguanosine