Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies.
We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP-enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.
Kranendonk
M
M
Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Vrije Universiteit van Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, Netherlands.
Carreira
F
F
Theisen
P
P
Laires
A
A
Fisher
C W
CW
Rueff
J
J
Estabrook
R W
RW
Vermeulen
N P
NP
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000494:pharmacology*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D009153:Mutagens; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase