Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450

Comparison of three types of expression systems

Michel Kranendonk, Charles W. Fisher, Rita Roda, Filipa Carreira, Patricia Theisen, Antonio Laires, J. Rueff, Nico P E Vermeulen, Ronald W. Estabrook

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.

Original languageEnglish
Pages (from-to)287-300
Number of pages14
JournalMutation Research - Genetic Toxicology and Environmental Mutagenesis
Volume439
Issue number2
DOIs
Publication statusPublished - 19 Feb 1999

Fingerprint

Cytochrome P-450 CYP1A2
NADPH-Ferrihemoprotein Reductase
Cytochrome P-450 Enzyme System
Escherichia coli
Oxidoreductases
Cytochrome Reductases
2-amino-3-methylimidazo(4,5-f)quinoline
Cytochromes
Electron-Transferring Flavoproteins
Aflatoxin B1
Liver
NADP
Rodentia

Keywords

  • Biotransformation
  • Chemical carcinogen
  • CYP1A2
  • E. coli
  • Human cytochrome P450 1A2
  • Mutagenicity
  • NADPH cytochrome P450 reductase

Cite this

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title = "Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: Comparison of three types of expression systems",
abstract = "Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.",
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Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 : Comparison of three types of expression systems. / Kranendonk, Michel; Fisher, Charles W.; Roda, Rita; Carreira, Filipa; Theisen, Patricia; Laires, Antonio; Rueff, J.; Vermeulen, Nico P E; Estabrook, Ronald W.

In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Vol. 439, No. 2, 19.02.1999, p. 287-300.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450

T2 - Comparison of three types of expression systems

AU - Kranendonk, Michel

AU - Fisher, Charles W.

AU - Roda, Rita

AU - Carreira, Filipa

AU - Theisen, Patricia

AU - Laires, Antonio

AU - Rueff, J.

AU - Vermeulen, Nico P E

AU - Estabrook, Ronald W.

PY - 1999/2/19

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KW - Chemical carcinogen

KW - CYP1A2

KW - E. coli

KW - Human cytochrome P450 1A2

KW - Mutagenicity

KW - NADPH cytochrome P450 reductase

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