RECOMBINANT CYP2E1 AMES MUTAGENICITY TEST

Todd D. Porter, Ph.D.

Cytochrome P450 2E1 (CYP2E1) is a heme monooxygenase that is involved in the metabolic activation of nitrosamines to electrophilic species that can react with DNA, resulting in DNA damage and mutation.¹  CYP2E1 is also responsible for the oxidation of many small lipophilic compounds, including many solvents and environmental contaminants.²  This enzyme is one of many cytochromes P450 found primarily in the liver, but also in other tissues including the lungs and kidneys.  The enzyme is found on the endoplasmic reticulum of the cell along with cytochrome P450 reductase, its obligate electron donor protein.  

Nitrosamines have long been recognized as carcinogens in mammals, but they have been unexpectedly poor mutagens in bacterial mutagenicity assays, such as the Ames test.³  A variety of explanations have been put forward over the years to explain this lack of correlation between mutagenicity and carcinogenicity, and have prompted the studies undertaken in my laboratory.⁴  

In a standard Ames assay a rat liver homogenate is used to provide the cytochromes P450 necessary for activation of mutagens to their reactive forms.  The suspected mutagen is mixed with the homogenate, where it is metabolized to its reactive form, and then an appropriate tester strain of Salmonella typhimurium is added to the mix.  The activated mutagen enters the bacterium and reacts with DNA to cause mutations; mutations in the operon for histidine biosynthesis that restore function ("reversions") allow the bacterium to grow on histidine-deficient medium, and result in visible colonies on the assay plate.  The greater the number of colonies, the more mutagenic the chemical.

We have taken the approach of placing the activating enzyme pathway inside the bacterial cell, and thereby eliminate the need for a rat liver homogenate.  Our tester strains coexpress human cytochrome P450 2E1 and cytochrome P450 reductase, and can directly activate mutagens that are P450 2E1 substrates, such as many nitrosamines.  The resulting test system shows greater sensitivity to these substances than does a standard Ames assay which uses a rat liver homogenate for activation.⁴

1. Yang CS, Yoo JS, Ishizaki H, Hong JY.  Cytochrome P450IIE1: roles in nitrosamine metabolism and mechanisms of regulation.  Drug Metab Rev 1990;22(2-3):147-59.  Nouso K, Thorgeirsson SS, Battula N.  Stable expression of human cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA.  Cancer Res 1992 Apr 1;52(7):1796-800 [abstract].  Yamazaki H, Oda Y, Funae Y, Imaoka S, Inui Y, Guengerich FP, Shimada T.  Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium
   strain (NM2009).  Carcinogenesis 1992 Jun;13(6):979-85 [abstract].  Kushida H, Fujita K, Suzuki A, Yamada M, Endo T, Nohmi T, Kamataki T.  Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase.  Carcinogenesis 2000 Jun;21(6):1227-32 [abstract]. 
2. Raucy JL, Kraner JC, Lasker JM.  Bioactivation of halogenated hydrocarbons by cytochrome P4502E1.  Crit Rev Toxicol 1993;23(1):1-20 [abstract].
3. McCann J, Choi E, Yamasaki E, Ames BN.  Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals.  Proc Natl Acad Sci U S A 1975 Dec;72(12):5135-9 [abstract].  McMahon RE, Cline JC, Thompson CZ.  Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens.  Cancer Res 1979 Mar;39(3):682-93 [abstract].  
4. Cooper MT, Porter TD.  Mutagenicity of nitrosamines in methyltransferase-deficient strains of salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase.  Mutat Res 2000 Nov 6;454(1-2):45-52 [abstract].

• Further information on our recombinant CYP2E1 Ames test
   
• Cytochrome b₅ coexpression increases the mutagenicity of nitrosamines