A NIH/3T3 cell line stably expressing human cytochrome P450-3A4 used in combination with a lacZ' shuttle vector to study mutagenicity.
An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ' gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B1 were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B1 increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B1. The system described in this paper opens the possibility to study the capacity of single human cytochrome P450s to activate xenobiotics into mutagenic metabolites. Since activation, phase II metabolism, DNA repair and an endpoint for mutations are all present in one cell, this system will be useful in screening as well as in mechanistic studies.
De Groene
E M
EM
Research Institute of Toxicology, Utrecht University, The Netherlands.
Seinen
W
W
Horbach
G J
GJ
D016604:Aflatoxin B1 / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005822:Genetic Vectors; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D010957:Plasmids / Q000502:physiology; D012313:RNA / Q000032:analysis; D013739:Testosterone / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D009153:Mutagens; D013739:Testosterone; D012313:RNA; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D005020:Ethyl Methanesulfonate; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human