Retroviral mediated expression of human cytochrome P450 2A6 in C3H/10T1/2 cells confers transformability by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
In order to develop more efficient in vitro systems for the study of pro-mutagenic or pro-carcinogenic chemicals, we have produced transgenic C3H/10T1/2 cell lines expressing human cytochrome P450 (CYP) 2A6. A retroviral vector containing the cDNA was packaged in psi-2 cells, and used to infect C3H/10T1/2 cells. From 100 G418-resistant clones initially isolated, three cell lines were chosen for further study based upon their morphologies, growth rates and CYP2A6-dependent coumarin 7-hydroxylase activities. Infected clone 10T1/2-04, like the 10T1/2 cells, had no detectable CYP2A6 enzyme activity, while clones 10T1/2-10 and 10T1/2-29 had microsomal CYP2A6 enzyme activities within the range found in human liver microsomes. CYP2A6 protein levels were in agreement with the observed enzyme activities. Southern blots revealed that cells from clone 10T1/2-04 contained a vector lacking the CYP2A6 cDNA, while cells from clones 10T1/2-10 and 10T1/2-29 contained multiple full-length inserts. Southern analysis also indicated the presence of an endogenous CYP2A6 ortholog in the four cell lines. All cell lines exhibited about equal sensitivity to induction of cytotoxicity and conversion to ouabain resistance by the direct acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine. The four lines were also about equally sensitive to transformation by benzo[a]pyrene, a chemical requiring metabolic activation. However, only clones 10T1/2-10 and 10T1/2-29, which express CYP2A6 activity, were mutated and morphologically transformed by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Tiano
H F
HF
National Institute of Environmental Health Sciences, Laboratory of Environmental Carcinogenesis and Mutagenesis, Research Triangle Park, NC 27709.
Hosokawa
M
M
Chulada
P C
PC
Smith
P B
PB
Wang
R L
RL
Gonzalez
F J
FJ
Crespi
C L
CL
Langenbach
R
R
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003001:Cloning, Molecular; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity*; D012190:Retroviridae / Q000235:genetics*
D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6