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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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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Basic Protocol 1: GC/MS Assay for Pyrimidopurinone

  • 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

  • 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

  • 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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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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