Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens.
In this study we describe the development of strain BMX100, a new Escherichia coli K12 tester strain, derived from MX100, a strain which was constructed for detection of mutagens and for mechanistic studies of chemical carcinogens. We demonstrate here that strain BMX100 can be used for stable expression of human CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochrome P450 reductase. Mutagenicity of precarcinogens known to be bioactivated by CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), could be detected. The mutagenic activity of 2-AA using BMX100 expressing CYP1A2 alone and in combination with rat CYP reductase was respectively 10 and 20 times higher than in BMX100 with the standard metabolic activation system, rat liver S9 fraction. Furthermore, the mutagenicity of 2-AA could be nullified by alpha-naphthoflavone, a known inhibitor of CYP1A2. IQ responded equally in BMX100 expressing the CYP1A2-reductase fusion protein as compared with usage of rat liver S9 fraction. Rat liver S9 fraction was much more potent in generating a mutagenic response to AFB1 in BMX100 than in the strain expressing human CYP1A2 alone or CYP1A2 fused to rat reductase. The results described in this study demonstrate that this new E.coli strain can function as a human CYP1A2-competent prokaryotic mutagenicity test system and they seem to characterize BMX100 as a strain of interest for studies to identify individual human CYPs involved in bioactivation and bioinactivation reactions of putative genotoxins.
Kranendonk
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Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Portugal.
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D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity; D016604:Aflatoxin B1 / Q000633:toxicity; D000818:Animals; D000873:Anthracenes / Q000633:toxicity; D001564:Benzo(a)pyrene / Q000633:toxicity; D001711:Biotransformation / Q000235:genetics; D002273:Carcinogens / Q000633:toxicity*; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D009152:Mutagenicity Tests; D011804:Quinolines / Q000633:toxicity; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000873:Anthracenes; D002273:Carcinogens; D011804:Quinolines; D011993:Recombinant Fusion Proteins; C029216:2-amino-3-methylimidazo(4,5-f)quinoline; D001564:Benzo(a)pyrene; D015112:4-Nitroquinoline-1-oxide; C006593:2-anthramine; D016604:Aflatoxin B1; D019388:Cytochrome P-450 CYP1A2