2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N-acetyltransferase 2.
Heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) are present in diet and cigarette smoke. Bioactivation in humans includes N-hydroxylation catalyzed by cytochrome P4501A2 possibly followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A2 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A2 catalytic activity levels did not differ significantly (P > 0.05) among the CYP1A2-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of N-acetyltransferase (P = 0.0001) and N-hydroxy-PhIP O-acetyltransferase (P = 0.0170) catalytic activity than cells transfected with NAT2*5B. PhIP caused dose-dependent decreases in cell survival and significant (P < 0.001) increases in mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in all the CYP1A2-transfected cell lines. Transfection with NAT2*4 or NAT2*5B did not further increase hprt mutagenesis. PhIP-induced hprt mutant cDNAs were sequenced, and 80% of the mutations were single base substitutions at G:C base pairs. dG-C8-PhIP DNA adduct levels were dose-dependent in the order: untransfected < transfected with CYP1A2 < transfected with CYP1A2 and NAT2*5B < transfected with CYP1A2 and NAT2*4. Following incubation with 1.2 microM PhIP, DNA adduct levels were significantly (P < 0.05) higher in CHO cells transfected with CYP1A2/NAT2*4 versus CYP1A2/NAT2*5B. These results strongly support an activation role for CYP1A2 in PhIP-induced mutagenesis and DNA damage and suggest a modest effect of human NAT2 and its genetic polymorphism on PhIP DNA adduct levels.
Metry
Kristin J
KJ
Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Zhao
Shuang
S
Neale
Jason R
JR
Doll
Mark A
MA
States
J Christopher
JC
McGregor
W Glenn
WG
Pierce
William M
WM
Hein
David W
DW
D000107:Acetylation; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002273:Carcinogens / Q000633:toxicity*; D006224:Cricetinae; D003412:Cricetulus; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000633:toxicity*
C502239:2-amino-1-methyl-6-phenolimidazo(4,5-b)pyridine-DNA adduct; D002273:Carcinogens; D018736:DNA Adducts; D007093:Imidazoles; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human