Mutagenicity of cytochrome P450 2E1 substrates in the Ames test with the metabolic competent S. typhimurium strain YG7108pin3ERb5.
Poor metabolic competence of in vitro systems was proposed to be one of their major shortcomings accounting for false negative results in genotoxicity testing. For several "low molecular weight cancer suspects" this was specifically attributed to the lack of cytochrome P450 2E1 (CYP2E1) in conventional in vitro metabolising systems. One promising attempt to overcome this problem is the transfection of "methyltransferase-deficient"S. typhimurium strains with the plasmid pin3ERb5. This plasmid contains DNA encoding for a complete electron transport chain, comprising P450 reductase, cytochrome b5 and cytochrome P450 2E1. In order to answer the question if CYP2E1 substrates that yield negative or inconclusive results in the Ames test can be activated by metabolic competent bacterial strains, we used YG7108pin3ERb5 to investigate the following compounds: acetamide, acrylamide, acrylonitrile, allyl chloride, ethyl acrylate, ethyl carbamate, methyl-methacrylate, vinyl acetate, N-nitrosopyrrolidine, trichloroethylene and tetrachloroethylene. N-Nitrosodiethylamine served as a positive control. In addition to these known or proposed CYP2E1 substrates, we investigated the polycyclic aromatic hydrocarbon benzo[alpha]pyrene and the heterocyclic aromatic amines 2-aminofluorene and 2-aminoanthracene.


RESULTS
The extensive metabolic competence of the transformed strain is underlined by results showing strong mutagenicity between 10 and 500 micro g N-nitrosopyrrolidine per plate. Unexpectedly, 2-aminoanthracene was mutagenic at a concentration range between 25 and 250 micro g per plate using YG7108pin3ERb5. Moreover, we demonstrate for the first time a clear response of sufficiently characterised allyl chloride in the Ames test at a reasonably low concentration range between 300 and 1500 micro g per plate. We achieved similar results in the parent strain YG7108 with conventional metabolic activation. Without metabolic activation less pronounced mutagenicity occurred, suggesting a contribution of a direct alkylating effect. Propylene oxide is usually contained in allyl chloride as stabilizer at amounts up to 0.09%. Though YG7108 revealed to be very sensitive towards propylene oxide, allyl chloride dissolved in water was not mutagenic, showing that no water soluble compounds contribute to its mutagenicity. None of the remaining compounds showed mutagenic effects using YG7108pin3ERb5.


CONCLUSIONS
YG7108pin3ERb5 and its parent strain YG7108 are sensitive for compounds which are negative in conventional tester strains including N-nitrosodiethylamine, N-nitrosopyrrolidine, propylene oxide and allyl chloride.
Emmert
Birgit
B
Department of Occupational and Social Health, Georg-August-University Göttingen, Waldweg 37, Göttingen, Germany. baut@gwdg.de
Bünger
Jürgen
J
Keuch
Kristian
K
Müller
Michael
M
Emmert
Steffen
S
Hallier
Ernst
E
Westphal
Götz A
GA
D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D004305:Dose-Response Relationship, Drug; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000145:classification / Q000633:toxicity*; D010957:Plasmids / Q000235:genetics; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D013379:Substrate Specificity; D014162:Transfection; D015262:Xenobiotics / Q000145:classification / Q000633:toxicity*
D004269:DNA, Bacterial; D009153:Mutagens; D015262:Xenobiotics; D019392:Cytochrome P-450 CYP2E1