Measurement of a Malondialdehyde‐DNA Adduct

John P. Plastaras1, Lawrence J. Marnett1

1 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.2
DOI:  10.1002/0471140856.tx0302s00
Online Posting Date:  May, 2001
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Abstract

Determining the levels of various DNA adducts has become an essential tool in understanding the toxicology of carcinogens. Direct measurement of DNA adduct levels, the true biologically effective dose of a mutagen, can be correlated with biological outcomes or used to probe mechanisms of adduct formation. Each adduct to be measured requires a specific assay. Malondialdehyde is a carcinogenic and mutagenic electrophile that is endogenously produced during peroxidation of polyunsaturated fatty acids. Its reaction with deoxyguanosine produces a fluorescent exocyclic pyrimidopurinone that can be detected by gas chromatographic/negative chemical ionization‐electron capture mass spectroscopy. Methods for preparing an immunoaffinity gel and for HPLC quatification of nucleosides are also included.

     
 
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Table of Contents

  • Basic Protocol 1: GC/MS Assay for Pyrimidopurinone
  • Support Protocol 1: Preparation of Anti‐M1G Immunoaffinity Gel
  • Support Protocol 2: HPLC Quantification of Nucleosides
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: GC/MS Assay for Pyrimidopurinone

  Materials
  • Tissue of interest
  • MOPS/sucrose buffer (see recipe), 4°C
  • 2× lysis buffer (Applied Biosystems)
  • 920 U/µl ribonuclease A (Sigma)
  • 100,000 U/µl ribonuclease T 1 (Boehringer Mannheim)
  • 10 to 20 U/µl proteinase K (Sigma)
  • Anti‐oxidant MOPS buffer (see recipe)
  • Phenol/chloroform/water reagent (Applied Biosystems)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate ( appendix 2A)
  • 100% and 70% (v/v) ethanol
  • 10 mM MOPS/100 mM NaCl, pH 7.0
  • 1.5 ng/ml [2D 2]M 1G‐dR standard (see Chaudhary et al., , for synthesis procedure, which is relatively straightforward)
  • Deoxyribonuclease (DNase) I stock solution (see recipe)
  • Nuclease P1 stock solution (see recipe)
  • 25 mM ZnCl 2
  • 0.4 M MOPS, pH 7.8
  • 20 U/µl alkaline phosphatase (Sigma)
  • Anti‐M 1G immunoaffinity gel (see protocol 2)
  • PBS ( appendix 2A)
  • Nondeuterated M 1G‐dR standard (see Chaudhary et al., )
  • Methanol
  • Nanopure water
  • Acetone
  • Concentrated formic acid (88% w/v)
  • recipePFB‐Br derivatization solution (see recipe)
  • Methylene chloride
  • N,O‐bis(Trimethylsilyl) trifluoroacetamide (BSTFA; Pierce)
  • Derivatized deuterated standard (see recipe)
  • Polytron homogenizer (Brinkmann)
  • Silica (Supelclean LC‐Si, Supelco)
  • 10‐µl Hamilton syringe
  • Repeater pipetter and a range of disposable pipets
  • Ultrafree‐Probind filters with modified PVDF membrane (Millipore)
  • End‐over‐end mixer
  • Solid‐phase extraction vacuum manifold
  • 6‐ml glass columns with Teflon frits (Supelco)
  • 5‐ml glass conical tubes with screw caps
  • 50‐ml syringe with column attachment for positive pressure
  • 60°C sand bath
  • Stills for drying solvents (see Figure .)
  • Vibrating shaker
  • Nitrogen evaporation chamber: N‐Evap (Organomation) or equivalent
  • Mortar and pestle
  • 0.5‐ or 1‐ml glass pipet and pipet pump
  • Hewlett Packard MS Engine (model HP5989A) equipped with Series II Plus gas chromatograph (model HP5890), autoinjector (model 7673 GC/SFC injector), electronic pressure programming, and the negative chemical ionization option (or equivalent GC/MS equipment)
  • Sample vials and inserts
  • Vial caps
  • Vial cap crimper
  • 50‐µl micropipets
  • Micropipetter

Support Protocol 1: Preparation of Anti‐M1G Immunoaffinity Gel

  Materials
  • 100 to 200 mg monoclonal anti‐M 1G IgG antibody, protein A purified (Oxford)
  • CNBr‐activated Sepharose 4B, freeze dried (Sigma)
  • 1 mM HCl
  • Coupling buffer: 0.1 M NaHCO 3/0.5 M NaCl, pH 8.3
  • Blocking buffer: 0.1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • Storage buffer: 0.1 M Tris⋅Cl/0.02% (w/v) sodium azide, pH 8.0
  • 0.1 M sodium acetate/0.5 M NaCl, pH 4.0
  • 200‐ml glass storage bottle

Support Protocol 2: HPLC Quantification of Nucleosides

  Materials
  • DNA hydrolysate (see protocol 1, step )
  • Solvent A: 50 mM ammonium acetate (pH 6.2), filtered and degassed
  • Solvent B: acetonitrile, filtered and degassed (solvents filtered with 0.2‐µm GH‐Polypro membrane; Gelman)
  • 1 mM deoxyribonucleoside mix (see recipe)
  • 4.6 mm × 25 cm octadecylsilyl (Ultrasphere ODS) HPLC column (Beckman)
  • HPLC system with ultraviolet detector
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Figures

Videos

Literature Cited

Literature Cited
   Agarwal, S. and Draper, H.H. 1992. Isolation of a malondialdehyde‐deoxyguanosine adduct from rat liver DNA. Free Radical Biol. Med. 13:695‐699.
   Basu, A.K. and Marnett, L.J. 1983. Unequivocal demonstration that malondialdehyde is a mutagen. Carcinogenesis 4:331‐333.
   Bernheim, F., Bernheim, M.L.C., and Wilbur, K.M. 1948. The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174:257‐264.
   Chaudhary, A.K., Nokubo, M., Marnett, L.J., and Blair, I.A. 1994a. Analysis of the malondialdehyde‐2′‐deoxyguanosine adduct in rat liver DNA by gas chromatography/electron capture negative chemical ionization mass spectrometry. Biol. Mass Spectrom. 23:457‐464.
   Chaudhary, A.K., Nokubo, M., Reddy, G.R., Yeola, S.N., Morrow, J.D., Blair, I.A., and Marnett, L.J. 1994b. Detection of endogenous malondialdehyde‐deoxyguanosine adducts in human liver. Science 265:1580‐1582.
   Diczfalusy, U., Falardeau, P., and Hammarstrom, S. 1977. Conversion of prostaglandin endoperoxides to C17‐hydroxyacids by human platelet thromboxane synthase. FEBS Letts. 84:271‐274.
   Fink, S.P., Reddy, G.R., and Marnett, L.J. 1997. Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci. U.S.A. 94:8652‐8657.
   Janero, D.R. 1990. Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biol. Med. 9:515‐540.
   Marnett, L.J., Basu, A.K., O'Hara, S.M., Weller, P.E., Rahman, A.F.M.M., and Oliver, J.P. 1986. Reaction of malondialdehyde with guanine nucleosides: Formation of adducts containing oxadiazabicyclononene residues in the base‐pairing region. J. Am. Chem. Soc. 108:1348‐1350.
   Mukai, F.H. and Goldstein, B.D. 1976. Mutagenicity of malondialdehyde, a decomposition product of peroxidized polyunsaturated fatty acids. Science 191:868‐869.
   Rouzer, C.A., Chaudhary, A.K., Nokubo, M., Ferguson, D.M., Reddy, G.R., Blair, I.A., and Marnett, L.J. 1997. Analysis of the malondialdehyde‐2'‐deoxyguanosine adduct, pyrimidopurinone, in human leukocyte DNA by gas chromatography/electron capture negative chemical ionization/mass spectrometry. Chem. Res. Toxicol. 10:181‐188.
   Seto, H., Okuda, T., Takesue, T., and Ikemura, T. 1983. Reaction of malondialdehyde with nucleic acid. I. Formation of fluorescent pyrimido[1,2‐a]purin‐10(3H)‐one nucleosides. Bull. Chem. Soc. Jpn. 56:1799‐1802.
   Sevilla, C.L., Mahle, N.H., Eliezer, N., Uzieblo, A., O'Hara, S.M., Nokubo, M., Miller, R., Rouzer, C.A., and Marnett, L.J. 1997. Development of monoclonal antibodies to the malondialdehyde‐deoxyguanosine adduct, pyrimidopurinone. Chem. Res. Toxicol. 10:172‐180.
   Spalding, J.W. 1988. Toxicology and carcinogenesis studies of malondialdehyde sodium salt (3‐hydroxy‐2‐propenal, sodium salt) in F344/N rats and B6C3F1 mice. NTP Technical Report 331:5‐13.
   Vaca, C.E., Fang, J‐L., Mutanen, M. and Valsta, L. 1995. 32P postlabeling determination of DNA adducts of malonaldehyde in humans: Total white blood cells and breast tissue. Carcinogenesis 16:1847‐1851.
   Yau, T.M. 1979. Mutagenicity and cytotoxicity of malondialdehyde in mammalian cells. Mech. Ageing Dev. 11:137‐144.
Key References
   Rouzer, C.A. 1997. See above.
  Original publication of GC/MS assay employing immunoaffinity chromatography step as well as measurement of adduct levels in human leukocyte DNA.
   Sevilla, C.L. 1997. See above.
  Description of anti‐M1G monoclonal antibody.
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