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 test er 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 fo ur new strains were compared for CYP- and RED-expression levels and CYP act ivities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CY P2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP 3A5 at intermediate expression levels. Membranes of all five tester bacteri a demonstrated similar RED-expression levels, except for the two CYP3A-cont aining bacteria which demonstrated slightly increased RED-levels. CYP-activ ities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), co umarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CY P3A5) activities. Reaction rates were comparable with those obtained previo usly for these CYP-enzymes, except for CYP3A5 which demonstrated a lower ac tivity. Benzo[ a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mut agenicity in the CYP1A1 expressing strain with mutagenic activities, respec tively, approximately 10-fold and 100-fold higher as compared with those ob tained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a s ignificant mutagenicity with all CYP expressing strains, albeit lower as co mpared to those obtained with the use of rat liver S9. CYP1A2 was approxima tely 3-fold more effective in generating a mutagenic response of AFB1 as co mpared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in A FB1 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. (C) 1999 Elsevier Science B.V. All rights r eserved.