MUTAGENICITY OF NITROSAMINES IN RECOMBINANT CYP2E1 SALMONELLA STRAINS 

Although nitrosamines are environmentally significant carcinogens, the use of short-term bioassays to identify the presence of these compounds and to assess their mutagenic potential has been problematic.  The Ames mutagenicity test is one such widely used bioassay, but the traditional Ames Salmonella typhimurium strains are largely insensitive to nitrosamines.  

Dialkylnitrosamines, the most commonly encountered nitrosamines, are metabolized by cytochrome P450 2E1 to methylating agents, which methylate DNA at the O⁶ and N⁷ positions of guanine.  DNA methylation causes base substitution mutations, but most bacteria are efficient at repair of these types of mutations.   Dr. T. Nohmi at the National Institute of Health Sciences, Japan, has developed several strains of Salmonella that are deficient in the repair of methylated nucleic acids in DNA.¹  These strains lack one or both of the known DNA repair methyltransferases: ogt, a constitutive O⁶-methylguanine transferase; and ada, a minor, weakly inducible  O⁶-methylguanine transferase.  Strain YG7104 lacks the ogt methyltransferase, while strain YG7108 lacks both methyltransferases.  These two strains, and especially YG7108, are highly sensitive to the mutagenic effects of methylating agents.  

We have used recombinant DNA techniques to place cDNA clones for human cytochrome P450 2E1 (CYP2E1) and its obligate electron transfer partner, cytochrome P450 reductase, on a plasmid (pIN3/2E1/OR) that we have transferred into the methyltransferase-deficient strains YG7104 and YG7108.²  The expression of CYP2E1 and reductase can be induced in the transformed cells by the addition of isopropylthiogalactoside (IPTG), and the cells then gain the ability to activate several dialkylnitrosamines to their reactive metabolites, as shown below for dimethylnitrosamine (DMN):

The high sensitivity of the YG strains to methylating agents is shown to the right, where DMN is activated by a rat liver S9 fraction.  S9 fraction contains CYP2E1 and P450 reductase, as well as many other rat enzymes, but activation takes place outside of the bacterial cell, and thus the activated mutagen must diffuse into the cell to react with DNA and cause mutations.  Strain TA1535, a traditional strain of Salmonella used for Ames testing, shows no sensitivity to nitrosamines.  YG7108, which lacks both methyltransferases, shows significantly better sensitivity than YG7104, which still contains an active ada methyltransferase.

When the CYP2E1 activating system is expressed inside the bacterial cell from the pIN3/2E1/OR plasmid a significant increase in sensitivity is obtained, as shown to the right.  Sensitivity in YG7108 is more than doubled; note, however, that TA1535 remains insensitive, indicating that its DNA repair system is highly efficient.

The recombinant YG strains also show good sensitivity to longer chain dialkylnitrosamines, such as diethylnitrosamine (DEN), dipropylnitrosamine (DPN), and dibutylnitrosamine (DBN).  However, the activity of CYP2E1 decreases as chain-length increases, such that DPN and DBN are relatively poor substrates.  These compounds also exhibit significant cytotoxicity at higher concentrations.  Although not shown, with longer chain nitrosamines the difference in sensitivity between YG7108 and YG7104 decreases, consistent with the specificity of the ada enzyme (absent in YG7108) for methyl and ethyl DNA adducts.³  

In summary, these recombinant strains are significantly more sensitive to nitrosamines than standard Ames strains in the presence of an external activating system (S9 fraction).  These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.  Further studies indicate that the addition of cytochrome b₅ to this coexpression system provides an additional increase in sensitivity in these strains.

1. Yamada M, Sedgwick B, Sofuni T, Nohmi T.  Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine.  J Bacteriol 1995 Mar;177(6):1511-9 [abstract]
2. 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]
3. Morimoto K, Dolan ME, Scicchitano D, Pegg AE.  Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli.  Carcinogenesis 1985 Jul;6(7):1027-31 [abstract]
   
   

For more information on the recombinant CYP2E1 Ames test:

• Cytochrome b₅ Coexpression Increases the Mutagenicity of Nitrosamines

Back to the Recombinant CYP2E1 Ames Mutagenicity Test page