Rat liver cytochrome P450IA2 synthesized by transfected COS-1 cells efficiently activates food-derived promutagens.
A standard calcium phosphate technique was used to obtain transient expression of cDNAs for rat liver cytochrome P450s in COS-1 cells. Cells transfected with a pMT2-based vector expressing P450IA2 cDNA (pMT2-IA2) had high acetanilide-4-hydroxylase activity and very low aryl hydrocarbon hydroxylase (AHH) activity. Cells transfected with a hybrid expression vector, pMT2-IA2/IA1, coding for a P450IA2/IA1 fusion protein (consisting of the amino-terminal region of P450IA2 and the central and carboxy-terminal regions of P450IA1) had high AHH activity. This result and other data indicate that the P450IA2/IA1 fusion protein has the substrate specificity of P450IA1. Extracts of cells transfected with pMT2-IA2 readily converted 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and related food-derived promutagens into mutagenic forms. Extracts of cells transfected with pMT2-IA2/IA1 showed efficient activation of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp P-2). To facilitate comparison of activities of P450s synthesized from cDNA expression vectors, the promutagen activation assays were carried out with limiting enzyme and saturating or nearly saturating substrate concentrations. The transient expression system described here uses a standard expression vector and requires only microgram quantities of cell extract protein for activation of food-derived promutagens such as MeIQ and Trp P-2. It will be useful for identifying P450s active in promutagen activation and for analyzing structure-function relationships of different P450 molecules.
Trottier
Y
Y
Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Canada.
Waithe
W I
WI
Anderson
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002243:Carbolines / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D005822:Genetic Vectors; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects*; D009153:Mutagens / Q000737:chemistry; D010957:Plasmids; D011804:Quinolines / Q000737:chemistry; D011810:Quinoxalines / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology*; D012486:Salmonella typhimurium; D014162:Transfection
D002243:Carbolines; D009153:Mutagens; D011804:Quinolines; D011810:Quinoxalines; D011993:Recombinant Fusion Proteins; C017821:2-amino-3-methyl-9H-pyrido(2,3-b)indole; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; D003577:Cytochrome P-450 Enzyme System; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline; C019132:3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole