CYTOCHROME b5 COEXPRESSION INCREASES THE MUTAGENICITY OF NITROSAMINES

 

Cytochrome b5 is a ubiquitous, 17 kDa electron transfer protein found bound to the endoplasmic reticulum in most tissues; in erythrocytes it exists in a soluble form and is involved in methemoglobin reduction.  This hemeprotein provides electron-reducing equivalents that support a number of metabolic reactions, including fatty acid desaturation and elongation, plasmalogen biosynthesis, sterol biosynthesis, and P450 monooxygenation by some isoforms of this enzyme.1

Addition of cytochrome b5 to recombinant cytochrome CYP2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro,2 although the mechanism by which b5 enhances P450 reactions is not clear.  One possibility is that cytochrome b5 stimulates CYP2E1-dependent reactions via donation of the second electron needed to drive the P450 monooxygenase cycle.  To determine if this enhancement could be observed with recombinant expression systems in vivo, we constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b5 in the Salmonella typhimurium strains lacking ogt and ada methyltransferases (YG7104, ogt-; and YG7108, ogt-, ada-) described earlier.  These studies have now been published in Mutation Research and are described below.3  

pIN3b5ER tricistronic expression plasmidThe tricistronic plasmid constructed to express cytochrome b5, cytochrome P450 2E1, and cytochrome P450 reductase, is shown to the right.  Placing the b5 cDNA ahead of the P450 and reductase cDNAs gave the best expression and activity.  In this plasmid each cDNA is separated by about 20 nucleotides, which includes a ribosome binding site.  Thus, although translation can initiate independently for each protein, it is likely that all three cDNAs are transcribed as a single mRNA and translated sequentially by ribosomes.  We were not able to accurately quantitate the levels of these proteins due to their low expression in these cells; however, immunoblotting and earlier studies indicate that all three are expressed at approximately equivalent levels.

 

  

Comparison of DMN Sensitivity Between Salmonella Strains

 

Comparison of DMN activation by Salmonella strains

The figure to the right shows the enhancement in sensitivity to nitrosamines obtained with the various bacterial strains and expression plasmid constructs.  TA1535 is the base Ames strain, and shows no sensitivity to dimethylnitrosamine (DMN) in the presence of S9 fraction from ethanol-treated rats.  YG7108, which lacks the two DNA-methyltransferase enzymes involved in DNA repair, shows reasonable sensitivity in the presence of the ethanol-induced S9 fraction, illustrating the important role DNA repair plays in preventing nitrosamine mutagenicity in Salmonella.  Coexpression of CYP2E1 and P450 reductase in this strain (YG7108ER) affords a 5-fold  increase in sensitivity, as noted on the previous page.   Coexpression of cytochrome b5 with CYP2E1 and P450 reductase, in YG7108b5ER,  dramatically increases the sensitivity of this strain to DMN, illustrating the role cytochrome b5 plays in vivo in enhancing CYP2E1 activity. 

 
Cytochrome b5 Coexpression Increases the Sensitivity to Other Dialkylnitrosamines
 

Activation of dialkylnitrosamines by YG7108b5ER

The sensitivity of the YG7108b5ER strain to other dialkylnitrosamines is also increased at least 10-fold when cytochrome b5 is present (compare this figure to the nitrosamine activation figure on the previous page).  CYP2E1 is most active with short-chain nitrosamines, such as DMN and diethylnitrosamine (DEN).  Longer chain dialkylnitrosamines such as dipropyl- and dibutyl-nitrosamine (DPN and DBN) are weaker mutagens, and are activated slightly more effectively by CYP2A6.4 

 

 

The mechanism by which cytochrome b5 increases the activity of CYP2E1 remains unclear, and studies are underway in my laboratory to characterize this effect.  Although it has been suggested that b5 donates the second electron to CYP2E1 during catalysis, structural and conformational effects that do not involve electron transfer have also been proposed.5  Despite the many years of research on these proteins this issue remains unresolved.  Current studies with b5 mutants that do not bind heme, and the addition of other electron transport proteins, such as cytochrome b5 reductase, may help elucidate the mechanism of this stimulation in vivo.

 
References
  1. Vergeres G, Waskell L.  Cytochrome b5, its functions, structure and membrane topology.  Biochimie 1995;77(7-8):604-20 [abstract]
  2. Patten CJ, Ning SM, Lu AY, Yang CS.  Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5.  Arch Biochem Biophys 1986 Dec;251(2):629-38 [abstract]; Wang MH, Patten CJ, Yang GY, Paranawithana SR, Tan Y, Yang CS.  Expression and coupling of human cytochrome P450 2E1 and NADPH-cytochrome P450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells.  Arch Biochem Biophys 1996 Oct 15;334(2):380-8 [abstract]; Patten CJ, Koch P.  Baculovirus expression of human P450 2E1 and cytochrome b5: spectral and catalytic properties and effect of b5 on the stoichiometry of P450 2E1-catalyzed reactions.  Arch Biochem Biophys 1995 Mar 10;317(2):504-13 [abstract]; Schenkman JB, Jansson I.  Interactions between cytochrome P450 and cytochrome b5.  Drug Metab Rev 1999 May;31(2):351-64. 
  3. Cooper MT, Porter TD.  Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium.  Mutat Res 2001 Dec 12;484(1-2):61-8 [abstract]
  4. 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]
  5. Tamburini PP, White RE, Schenkman JB.  Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.  J Biol Chem 1985 Apr 10;260(7):4007-15 [abstract]; Yamazaki H, Johnson WW, Ueng YF, Shimada T, Guengerich FP.  Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.  J Biol Chem 1996 Nov 1;271(44):27438-44 [abstract]. 
     
 

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Comments to Todd D. Porter, Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082.  Phone 859 257-1137; FAX 859 257-7564
Last Modified: January 13, 2002
Copyright © 2001, University of Kentucky Chandler Medical Center