Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium.
Addition of cytochrome b(5) to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro. To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b(5) in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt(-); and YG7108, ogt(-), ada(-)). These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction). The four-fold increase in mutagenicity in the presence of cytochrome b(5) was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1. The greatest enhancement was obtained with a tricistronic construct in which the b(5) cDNA preceded the P450 and reductase cDNAs; placing the b(5) cDNA after the reductase cDNA was substantially less effective. These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.
Cooper
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Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536-0305, USA.
Porter
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D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008780:Methyltransferases; D009152:Mutagenicity Tests; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism / Q000633:toxicity*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009153:Mutagens; D009602:Nitrosamines; D011994:Recombinant Proteins; D014157:Transcription Factors; D015786:Cytochromes b5; D019392:Cytochrome P-450 CYP2E1; D009251:NADPH-Ferrihemoprotein Reductase; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; C067511:Ogt protein, E coli