Aldehyde Reduction by Cytochrome P450

Immaculate Amunom1, Sanjay Srivastava2, Russell A. Prough3

1 XenoTech LLC, Lenexa, Kansas, 2 Department of Medicine, Division of Cardiovascular Medicine, University of Louisville School of Medicine, Louisville, Kentucky, 3 Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.37
DOI:  10.1002/0471140856.tx0437s48
Online Posting Date:  May, 2011
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Abstract

This protocol describes the procedure for measuring the relative rates of metabolism of the α,β‐unsaturated aldehydes 9‐anthracene aldehyde (9‐AA) and 4‐hydroxy‐trans‐2‐nonenal (4‐HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9‐AA (a model α,β‐unsaturated aldehyde) by CYPs was adapted from an assay for 9‐anthracene oxidation published by Marini et al. (2003). For reduction of the endogenous aldehyde 4‐HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH, and the metabolites were separated by HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9‐AA and 4‐HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9‐AA) or HPLC coupled with a radiometric assay (4‐HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450−catalyzed azo reduction reactions. Curr. Protoc. Toxicol. 48:4.37.1‐4.37.15. © 2011 by John Wiley & Sons, Inc.

Keywords: Cytochrome P450; reduction reactions; α,β‐unsaturated aldehydes; inhibitors 4‐hydroxy‐2‐nonenal; 9‐anthracene aldehyde

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolism of Model α,β‐Unsaturated Aldehyde: 9‐Anthracene Aldehyde
  • Basic Protocol 2: Metabolism of Endogenous Aldehyde: 4‐Hydroxy‐trans‐2‐Nonenal
  • Basic Protocol 3: Identification of Metabolites by GC/MS
  • Basic Protocol 4: Analysis of Reduction Under Conditions of Anaerobiosis or Carbon Monoxide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Metabolism of Model α,β‐Unsaturated Aldehyde: 9‐Anthracene Aldehyde

  Materials
  • Microsomal fractions, expressed CYP fractions, or purified CYP
  • Phosphate/EDTA buffer, pH 7.4 (see recipe)
  • 2.5 mM 9‐anthracene aldehyde (9‐AA or 9‐anthraldehyde; Fluka 10603, Sigma‐Aldrich) in dimethylsulfoxide (DMSO) or ethanol
  • NADPH‐regenerating solution (see recipe)
  • 10 mM NADPH or NADP+stock solution (Sigma‐Aldrich; store up to 1‐2 weeks at 4°C)
  • 0.5 N NaOH (Sigma‐Aldrich)
  • Ethyl acetate (Thermo Fisher Scientific)
  • 0.5 N HCl
  • 1 mM 9‐anthracene carboxylic acid (9‐ACA, Aldrich S59065, Sigma‐Aldrich) in DMSO
  • 1 mM 9‐(hydroxymethyl)anthracene (9‐A‐MeOH or 9‐anthracene methanol, Aldrich 187240, Sigma‐Aldrich) in DMSO
  • 15‐ml screw‐cap conical tubes (polypropylene or borosilicate; e.g., Thermo Fisher Scientific)
  • 37°C shaking water bath
  • Low‐speed benchtop centrifuge (optional)
  • 3‐ml quartz fluorometer cuvette (several vendors)
  • Spectrofluorometer (e.g., Model LS50B, Perkin‐Elmer)
NOTE: 9‐AA, 9‐ACA, and 9‐A‐MeOH are light sensitive and should be kept in a brown bottle or wrapped in aluminum foil. When not in use, they can be stored at 4°C.

Basic Protocol 2: Metabolism of Endogenous Aldehyde: 4‐Hydroxy‐trans‐2‐Nonenal

  Materials
  • Microsomal protein or recombinant cytochrome P450
  • Phosphate/EDTA buffer, pH 7.4 (see recipe)
  • 2 mM [4‐3H]HNE stock solution: 2 mM non‐radioactive 4‐HNE (32100, Cayman Chemical) with sufficient radiolabeled 4‐HNE (Chandra and Srivastava, ; Srivastava et al., ) to give a stock of ∼0.2‐1.0 Ci/mmol
  • 10 mM NADPH stock solution (N5130, Sigma‐Aldrich; store up to 1‐2 weeks at 4°C)
  • Dry ice or liquid nitrogen
  • 15% (w/v) trichloroacetic acid (TCA, Sigma‐Aldrich)
  • 0.1% (v/v) trifluoroacetic acid (TFA, 302031, Sigma‐Aldrich)
  • HPLC‐grade water (high‐quality water from local source or purchased from Thermo Fisher Scientific or Burdick & Jackson solvents from VWR)
  • HPLC‐grade acetonitrile (Thermo Fisher Scientific or Burdick & Jackson solvents from VWR)
  • 15‐ml screw‐cap conical tubes (polypropylene or borosilicate; e.g., Thermo Fisher Scientific)
  • 37°C shaking water bath
  • Benchtop or high‐speed centrifuge
  • Scintillation counter (e.g., Packard Tricarb 2100TR, Packard Instrument Co.) and scintillation fluid (e.g., Ultimagold, Packard Instrument Co.)
  • HPLC system with:
    • Automated gradient controller with dual model 510 pumps (Waters)
    • Manual injector (2‐ml loop; Waters)
    • C18 reversed‐phase column (5 µm, 250 × 4.6 mm, Varian)
    • Radioactivity detector (Radiometric Flo‐one ß model A‐515, Packard Instrument Co.) or fraction collector (Model 2210, Bio‐Rad)

Basic Protocol 3: Identification of Metabolites by GC/MS

  Materials
  • HPLC fractions from [4‐3H]HNE reaction (see protocol 2)
  • Standards
  • Pentafluorobenzyl hydroxylamine (PFBHA, 76735, Sigma‐Aldrich)
  • Methanol (Thermo Fisher Scientific)
  • Hexane (Thermo Fisher Scientific or Burdick & Jackson solvents from VWR)
  • Nitrogen
  • N,O‐Bis(trimethylsilyl) trifluoroacetamide (BSTFA, Fluka 15222, Sigma‐Aldrich)
  • Helium carrier gas for gas chromatograph
  • Gas chromatograph (e.g., Agilent 6890/5973 GC/MS system, Agilent Technologies) under 70 eV electron ionization conditions
  • Bonded‐phase capillary column (DB‐5MS, 30 m × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific, Agilent)

Basic Protocol 4: Analysis of Reduction Under Conditions of Anaerobiosis or Carbon Monoxide

  Materials
  • Argon or carbon monoxide (commercial gas cylinder with regulator)
  • Oxygen‐scavenging and NADPH‐regenerating solution (see recipe)
  • 50‐ml Erlenmeyer flask
  • Serum stopper for Erlenmeyer flask with 13.88‐ or 15.85‐mm i.d. (14‐126BB or 14‐126DD; Thermo Fisher Scientific)
  • 1‐ml tuberculin syringes with 18‐G needles (Thermo Fisher Scientific)
  • Tygon tubing (Thermo Fisher Scientific)
  • Additional reagents and equipment for analysis of 9‐AA or 4‐HNE metabolism (see Basic Protocols protocol 11 and protocol 22)
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Figures

Videos

Literature Cited

   Amunom, I., Stephens, L.J., Tamasi, V., Cai, J., Pierce, W.M. Jr., Conklin, D.J., Bhatnagar, A., Srivastava, S., Martin, M.V., Guengerich, F.P., and Prough, R.A. 2007. Cytochromes P450 catalyze oxidation of α,β‐unsaturated aldehydes. Arch. Biochem. Biophys. 464:187‐196.
   Chandra, A. and Srivastava, S.K. 1997. A synthesis of 4‐hydroxy‐2‐trans‐nonenal and 4‐(3H) 4‐Hydroxy‐2‐trans‐nonenal. Lipids 32:779‐782.
   Fuller, A.T. 1937. Is p‐aminoazobenzenesulfonamide the active agent in prontosil therapy? Lancet i:94‐198.
   Guengerich, F.P. 1983. Oxidation‐reduction properties of rat liver cytochromes P‐450 and NADPH‐cytochrome P‐450 reductase related to catalysis in reconstituted systems. Biochemistry 22:2811‐2820.
   Guengerich, F.P. 2001. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol. 14:611‐650.
   Guengerich, F.P. 2007. Mechanisms of cytochrome P450 substrate oxidation: MiniReview. J. Biochem. Mol. Toxicol. 21:163‐168.
   Guengerich, F.P. 2008. Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 21:70‐83.
   Havemeyer, A., Grunewald, S., Wahl, B., Bittner, F., Mendel, R., Erdelyi, P., Fischer, J., and Clement, B. 2010. Reduction of N‐hydroxy‐sulfonamides, including N‐hydroxy‐valdecoxib, by the molybdenum‐containing enzyme MARC. Drug Metab. Dispos. 38:1917‐1921.
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   Marini, S., Grasso, E., Longo, V., Puccini, P., Riccardi, B., and Gervasi, P.G. 2003. 4‐Biphenylaldehyde and 9‐anthraldehyde: Two fluorescent substrates for determining P450 enzyme activities in rat and human. Xenobiotica 33:1‐11.
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   Milton, M.N., Elcombe, C.R., and Gibson, G.G. 1990. On the mechanism of induction of microsomal cytochrome P450IVA1 and peroxisome proliferation in rat liver by clofibrate. Biochem. Pharmacol. 40:2727‐2732.
   Prough, R.A., Coomes, M.W., Cummings, S.W., and Wiebkin, P. 1981. Metabolism of procarbazine [N‐isopropyl‐α‐(2‐methylhydrazino)‐p‐toluamide HCl]. Adv. Exp. Med. Biol. 136B:983‐996.
   Prough, R.A., Amunom, I., Tamasi, V., Conklin, D.J., Bhatnagar, A., and Srivastava, S. 2009. Cytochrome P450s catalyze reduction of lipid aldehydes metabolites. 16th North American Meeting of the International Society for the Study of Xenobiotics, Baltimore, Md., October 18–21, 2009, Abstract 87.
   Raza, H. and Levine, W.G. 1986a. Azoreduction of N,N‐dimethyl‐4‐aminoazobenzene (DAB) by rat hepatic microsomes. Selective induction by clofibrate. Drug Metab. Dispos. 14:19‐24.
   Raza, H. and Levine, W.G. 1986b. Effect of phenobarbital and β‐naphthoflavone on oxidative metabolism of N,N‐dimethyl‐4‐aminoazobenzene by regenerating rat‐liver microsomes and its response to sulphydryl compounds. Xenobiotica 16:827‐837.
   Shou, M., Lu, T., Krausz, K.W., Sai, Y., Yang, T., Korzekwa, K.R., Gonzalez, F.J., and Gelboin, H.V. 2000. Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism. Eur. J. Pharmacol. 394:199‐209.
   Srivastava, S., Ramana, K.V., Bhatnagar, A., and Srivastava, S.K. 2010. Synthesis, quantification, characterization, and signaling properties of glutathionyl conjugates of enals. Methods Enzymol. 474:297–313.
   Trefouel, J., Trefouel, J., Nitti, F.., and Bovet, D. 1935. Activite du p‐aminophenylsulfamide sur les infections streptococciques. Experimentales de la souris et du lapin. Comptes Rendus Seances Societe Biology 120:756‐762.
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Internet Resources
   http://www.bdbiosciences.com/research/admetox/services/discovery_service.jsp
  Several kits are available for measurement of various cytochromes P450 and various phase II enzyme reactions, including specific substrates and inhibitors. There are also websites that provide information about drugs that serve as P450 inhibitors. Two internet resources for specific inhibitors include psychresidentonline.com and BD GenTest.
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