Methods for Measuring DNA Adducts and Abasic Sites II: Methods for Measurement of DNA Adducts

James A. Swenberg1, Amy‐Joan L. Ham1, Hasan Koc1, David K. La1, Eric J. Morinello1, Brian F. Pachkowski1, Asoka Ranasinghe1, Patricia B. Upton1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.9
DOI:  10.1002/0471140856.tx0309s12
Online Posting Date:  August, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit contains protocols for analyzing DNA adducts separated from the DNA backbone. HPLC is used to quantify total guanine or ribo‐ or deoxynucleotides as well as methods for analyzing specific adducts. These methods include HPLC with electrochemical detection, immunoaffininty chromatography to enrich for specific adducts, and gas and liquid chromatography in combination with HPLC and mass spectrometry.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Measurement of Nucleic Acids by HPLC
  • Basic Protocol 1: HPLC Determination of Purine Bases
  • Basic Protocol 2: HPLC of Ribonucleoside and Deoxynucleoside Digests
  • Basic Protocol 3: HPLC‐ECD Analysis of 8‐OH‐dG
  • Support Protocol 1: Enzymatic DNA Digestion
  • Basic Protocol 4: HPLC‐ECD Analysis of N7‐Methylguanine
  • Basic Protocol 5: Quantification of N2,3‐Ethenoguanine by IA/GC/ECNCI/HRMS
  • Support Protocol 2: Preparation of Immunoaffinity (IA) Columns for DNA Adduct Purification
  • Support Protocol 3: Determination of Recovery and Adduct Purification
  • Basic Protocol 6: GC/ECNCI/HRMS Analysis of N2,3‐ethenoguanine (ɛGua), 7‐(hydroxyethyl) Guanine (7‐HEG), and 7‐(hydroxypropyl) Guanine (7‐HPG) Using a VG 70‐250SEQ Mass Spectrometer
  • Basic Protocol 7: Quantification of N7‐Guanine Adducts by LC/ESI/IDMS/MS
  • Basic Protocol 8: 32P‐Postlabeling Analysis of DNA Adducts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: HPLC Determination of Purine Bases

  Materials
  • Hydrated DNA solution
  • 0.1 N HCl
  • Mobile phase: 0.1 M ACS‐grade ammonium formate (pH 2.8)/10% HPLC‐grade methanol
  • Guanine (or adenine) standard for calibration curve
  • 22‐G needle
  • 70°C water bath or equivalent
  • 0.45‐µm filter
  • HPLC system:
  •  Isocratic HPLC pump
  •  500‐µl injection loop
  •  10‐µm × 25‐cm × 0.46‐cm Whatman Partisil 10 SCX analytical column
  •  Single‐wavelength UV detector set to 254 nm
  • Syringe and 22‐G needle
  • Additional reagents and equipment for determination of DNA concentration by spectrophotometric absorption (unit 3.8)

Basic Protocol 2: HPLC of Ribonucleoside and Deoxynucleoside Digests

  Materials
  • Mobile phase A: 50 mM NaH 2PO 4 (pH 5.5)/10% methanol
  • Mobile phase B: 90% methanol
  • Deoxyguanosine (dGuo), deoxyadenosine (dAdo), guanosine (rGuo), and adenosine (rAdo)
  • 50 mM NaH 2PO 4, pH 7.4 (optional)
  • 100 mM Bis‐Tris (pH 7.1)/100 mM MgCl 2
  • 100 mM Tris⋅Cl, pH 8.5 ( appendix 2A)
  • Sample digest (see protocol 4)
  • 0.45‐µm filter
  • HPLC system:
  •  500‐µl injection loop
  •  5‐µm × 25‐cm × 0.46‐cm reversed‐phase column
  •  Single‐wavelength UV detector set to 254 nm
  •  Isocratic HPLC pump

Basic Protocol 3: HPLC‐ECD Analysis of 8‐OH‐dG

  Materials
  • DNA sample (see protocol 4)
  • Mobile phase: 50 mM KH 2PO 4, (pH 5.5)/10% HPLC‐grade methanol
  • 8‐OH‐dG standard (see recipe)
  • HPLC system:
  •  100‐µl injection loop
  •  Electrochemical detector (i.e., E.S.A. Model 5600 CoulArray and Model 5040 cell with platinum target)
  •  25‐cm × 4.6‐mm Beckman Ultrasphere (or equivalent) C 18 column with 5‐µm particle size, room temperature
  • 37°C incubator
  • Additional reagents and equipment for HPLC determination of deoxyguanosine (see protocol 1)
NOTE: All water used must be distilled and deionized.

Support Protocol 1: Enzymatic DNA Digestion

  Materials
  • Digestion enzymes:
  •  200 U/0.1ml deoxyribonuclease I (Sigma‐Aldrich)
  •  0.1 U/0.1 ml spleen phosphodiesterase (Sigma‐Aldrich)
  •  0.5 U/0.1 ml snake venom phosphodiesterase (Worthington Biochemicals)
  •  10 U/0.1 ml alkaline phosphatase (Sigma‐Aldrich)
  • 10× digestion buffer: 400 mM Tris⋅Cl, pH 8.5 ( appendix 2A)/100 mM MgCl 2; store up to 1 month at 4°C
  • DNA sample

Basic Protocol 4: HPLC‐ECD Analysis of N7‐Methylguanine

  Materials
  • DNA sample
  • 1 M HCl, ice cold
  • Mobile phase, cold: 50 mM KH 2PO 4 (pH 5.5)/2% (v/v) HPLC‐grade methanol
  • 1 M NaOH
  • N7‐MG standards (see recipe)
  • HPLC system:
  •  100‐µl injection loop
  •  Electrochemical detector (E.S.A. Model 5600 CoulArray and Model 5040 cell  with platinum target)
  •  25 cm × 4.6–mm C 18 column with 5‐µm particle size, room temperature
  • 100°C water bath or equivalent
NOTE: All water used must be distilled and deionized.

Basic Protocol 5: Quantification of N2,3‐Ethenoguanine by IA/GC/ECNCI/HRMS

  Materials
  • DNA sample
  • 1 N HCl: dilute 3.3 ml concentrated HCl to 40 ml with water; store up to 6 months at room temperature
  • ∼2 fmol/ml [13C 4,15N 2]N2,3‐ɛGua internal standard
  • ∼1 fmol/ml N2,3‐ɛGua standard
  • 500 mM sodium phosphate buffer, pH 7.2 (see recipe; also see appendix 2A)
  • Immunoaffinity (IA) columns made with polyclonal antibodies to N2,3‐ɛGua (see protocol 7)
  • 5% (v/v) and 100% HPLC grade methanol
  • PBS/azide (see recipe)
  • 0.1 M formic acid
  • Potassium carbonate: grind to a fine powder with mortar and pestle and store at 60°C
  • Acetone, HPLC‐grade (≥99.9%)
  • 5% PFBBr (see recipe)
  • Hexane
  • Silica gel slurry: silica gel 60, 70‐230 mesh (Fisher) in hexane
  • Ethyl acetate, GC/GC‐MS grade
  • Dichloromethane, ACS or HPLC grade
  • 5% (v/v) ethyl acetate in hexane
  • 99.8% toluene
  • Helium gas
  • Methane gas
  • 22‐G needle
  • 70°C water bath or heating block with H 2O in the test‐tube holes
  • Centricon 10 concentrators
  • 13 × 100–mm silanized, disposable, borosilicate culture tubes
  • 5.75‐in. silanized Pasteur pipets
  • Silanized glass wool
  • 200‐µl genomic and 100‐µl gel‐loading aerosol‐resistant pipet tips
  • 1.8‐ml GC autosampler vials with inserts and caps
  • Silanized culture tubes with caps
  • Transfer pipets
  • GC‐MS system:
  •  Electron capture negative chemical ionization detector
  •  DB‐5MS 30‐m × 0.32‐mm × 0.1‐mm film column or equivalent
  •  Uniliner HP injection sleeve, 4.0 mm i.d. × 6.3 mm o.d. × 78.5 mm (Restek)
  • Additional reagents and equipment for quantifying DNA by UV spectroscopy (unit 3.8) and measurement of guanine by HPLC (see protocol 1)
NOTE: All hexane used in the protocol must be capillary GC/GC‐MS grade.

Support Protocol 2: Preparation of Immunoaffinity (IA) Columns for DNA Adduct Purification

  Materials
  • Protein A–Sepharose CL4B (Amersham Pharmacia Biotech)
  • 100 mM Tris⋅Cl, pH 7.4 ( appendix 2A)
  • Purified antibody, hybridoma cell supernatant, or antiserum against adduct of interest
  • 200 mM triethanolamine, pH 8.2: 13.3 ml in 500 ml (total) water; store up to 1 month at 4°C
  • 20 mM dimethylpimelidate (DMP; Pierce Chemical) in 200 mM triethanolamine, pH 8.2, fresh
  • 20 mM ethanolamine (Fisher Scientific) in 200 mM triethanolamine, pH 8.2
  • PBS/azide (see recipe)
  • 100% HPLC‐grade methanol (J.T. Baker or equivalent)
  • 12‐ml polyethylene minisorb tubes (Nunc) or 15‐ml centrifuge tubes
  • End‐over‐end mixer
  • Four 5‐ml disposable polystyrene columns with two frits each (Pierce Chemical)

Support Protocol 3: Determination of Recovery and Adduct Purification

  Materials
  • IA columns (see protocol 7)
  • 5% (v/v) and 100% methanol
  • PBS/azide (see recipe)
  • Neutralized standard (e.g., ∼1 fmol/ml N2,3‐ɛGua, pH 7.0) or sample
  • DNA sample
  • 0.1 M formic acid
  • Internal standard (optional)
  • Additional reagents and equipment for quantifying samples by HPLC (see protocol 1)

Basic Protocol 6: GC/ECNCI/HRMS Analysis of N2,3‐ethenoguanine (ɛGua), 7‐(hydroxyethyl) Guanine (7‐HEG), and 7‐(hydroxypropyl) Guanine (7‐HPG) Using a VG 70‐250SEQ Mass Spectrometer

  Materials
  • 99.999% helium (Sunox)
  • Perflurokerosine (PFK; PCR) or perfluorotributylamine (FC43; PCR)
  • Sample in toluene
  • HP‐5890 Series II gas chromatograph (GC) with appropriate column and 4.0 mm‐i.d. × 6.3‐mm‐o.d. × 78.5‐mm Restek uniliner coupled in‐line to a VG 70‐250SEQ mass spectrometer (Micromass UK) with EI/CI ion source and oscilloscope

Basic Protocol 7: Quantification of N7‐Guanine Adducts by LC/ESI/IDMS/MS

  Materials
  • DNA sample
  • Isotopically labeled internal standard (e.g., 13C 4‐N7‐HEG)
  • 100% methanol
  • SPE reversed‐phase cartridge, preferably polar endcapped (e.g., ODS‐AQ, Waters Chromatography or Aquasil, Keystone Scientific)
  • Calf thymus DNA
  • 22‐G needles
  • 95° and ∼60°C water baths
  • Centricon 30 filters
  • Autosampler vial with 250‐µl insert
  • Mass spectrometer with electrospray ionization source and the capability to conduct selected reaction monitoring (e.g., Thermoquest TSQ 7000, Micromass Quattro II)

Basic Protocol 8: 32P‐Postlabeling Analysis of DNA Adducts

  Materials
  • 100% and 50% methanol
  • DNA sample
  • Hydrolysis buffer (see recipe)
  • 0.24 U/µl micrococcal nuclease (Sigma‐Aldrich): aliquot and freeze up to 1 year at −80°C
  • 2 mg/ml spleen phosphodiesterase (Sigma‐Aldrich), aliquot and freeze up to 1 year at −80°C
  • 0.3 mM ZnCl 2
  • 0.25 M sodium acetate, pH 5
  • 5 mg/ml nuclease P1 (Sigma‐Aldrich): aliquot and freeze up to 1 year at −80°C
  • 0.5 M Tris base
  • Kinase buffer (see recipe)
  • 30 U/µl polynucleotide kinase (United States Biochemical): store up to 1 year at −20°C
  • 160 mCi/ml [γ‐32P]ATP (7000 Ci/mmol; ICN)
  • 100 mM Bicine, pH 9.6
  • 20 U/µl apyrase (Sigma‐Aldrich): aliquot and freeze up to 1 year at −80°C
  • 1 M and 1.7 M NaH 2PO 4, pH 6.0 ( appendix 2A)
  • 5.3 M lithium formate/8.5 M urea, pH 3.5
  • 1.2 M LiCl/0.5 M Tris⋅Cl (pH 8.0; appendix 2A)/8.5 M urea
  • 0.25 M LiCl
  • HPLC‐grade H 2O
  • 13 × 20–cm PEI cellulose TLC plates, flexible
  • 5 × 20–cm Whatman 17 wick
  • Blow dryer
  • 2.5‐cm Whatman no. 1 filter paper wick
  • Phosphorescent pen
  • Incubator or heating block
  • X‐ray film cassette
  • Darkroom
  • Liquid scintillation counter
CAUTION: When working with radioactivity, use appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out experiments and dispose of wastes in appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A). All work with radioactivity must be performed using acrylic shielding and monitored using a Geiger counter.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Achilli, G., Cellerino, G.P., Gamache, P.H., and Melzi d'Eril, G.V. 1993. Identification and determination of phenolic constituents in natural beverages and plant extracts by means of a coulometric electrode array system. J. Chromatogr. 632:111‐117.
   Ames, B.N. 1983. Dietary carcinogens and anticarcinogens. Science. 221:1256‐1264.
   Barbin, A., Laib, R.J., and Bartsch, H. 1985. Lack of miscoding properties of 7‐(2‐oxoethyl)guanine, the major vinyl chloride‐DNA adduct. Cancer Res. 45:2440‐2444.
   Bartsch, H., Barbin, A., Marion, M.J., Nair, J., and Guichard, Y. 1994. Formation, detection, and role in carcinogenesis of ethenobases in DNA. Drug Metab. Rev. 26:349‐371.
   Basu, A.K., Wood, M.L., Niedernhofer, L.J., Ramos, L.A., and Essigmann, J.M. 1993. Mutagenic and genotoxic effects of three vinyl chloride‐induced DNA lesions: 1,N6‐ethenoadenine, 3,N4‐ethenocytosine, and 4‐amino‐5‐(imidazol‐2‐yl)imidazole. Biochemistry 32:12793‐12801.
   Beranek, D.T., Weis, C.C., and Swenson, D.H. 1980. A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis 1:595‐606.
   Bianchini, F. and Wild, C.P. 1994. 7‐Methyldeoxyguanosine as a marker of exposure to environmental methylating agents. Toxicol. Lett. 72:175‐184.
   Boucheron, J.A., Richardson, F.C., Morgan, P.H., and Swenberg, J.A. 1987. Molecular dosimetry of O4‐ethyldeoxythymidine in rats continuously exposed to diethylnitrosamine. Cancer Res. 47:1577‐1581.
   Calléja, F., Jansen, J.G., Vrieling, H., Laval, F., and van Zeeland, A.A. 1999. Modulation of the toxic and mutagenic effects induced by methyl methanesulfonate in Chinese hamster ovary cells by overexpression of the rat N‐alkylpurine‐DNA glycosylase. Mutat. Res. 425:185‐194.
   Chen, H.J. and Chung, F.L. 1994. Formation of etheno adducts in reactions of enals via autoxidation. Chem. Res. Toxicol. 7:857‐860.
   Cheng, K.C., Preston, B.D., Cahill, D.S., Dosanjh, M.K., Singer, B., and Loeb, L.A. 1991. The vinyl chloride DNA derivative N2,3‐ethenoguanine produces G‐>A transitions in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 88:9974‐9978.
   Chetsanga, C.J., Lozon, M., Makaroff, C., and Savage, L. 1981. Purification and characterization of Escherichia coli formamidopyrimidine‐DNA glycosylase that excises damaged 7‐methylguanine from deoxyribonucleic acid. Biochemistry 20:5201‐5207.
   de Groot, A.J.L., Jansen, J.G., van Valkenburg, C.F.M., and van Zeeland, A.A. 1994. Molecular dosimetry of 7‐alkyl‐ and O6‐alkylguanine in DNA by electrochemical detection. Mutat. Res. 307:61‐66.
   Dunn, B.P. and San, R.H.C. 1988. HPLC enrichment of hydrophobic DNA‐carcinogen adducts for enhanced sensitivity of 32P‐postlabeling analysis. Carcinogenesis 9:1055‐1060.
   Fasman, G.D. (ed.) 1976. Handbook of Biochemistry and Molecular Biology, Third Edition, pp. 270‐273. CRC Press, Cleveland.
   Fedtke, N., Boucheron, J.A., Walker, V.E., and Swenberg, J.A. 1990. Vinyl chloride‐induced DNA adducts. II: Formation and persistence of 7‐(2′‐oxoethyl)guanine and N2,3‐ethenoguanine in rat tissue DNA. Carcinogenesis 11:1287‐1292.
   Friesen, M.D., Garren, L., Prevost, V., and Shuker, D.E. 1991. Isolation of urinary 3‐methyladenine using immunoaffinity columns prior to determination by low‐resolution gas chromatography‐mass spectrometry. Chem. Res. Toxicol. 4:102‐106.
   Froment, O., Boivin, S., Barbin, A., Bancel, B., Trepo, C., and Marion, M.J. 1994. Mutagenesis of ras proto‐oncogenes in rat liver tumors induced by vinyl chloride. Cancer Res. 54:5340‐5345.
   Garland, W.A. and Powell, M.L. 1981. Quantitative selected ion monitoring (QSIM) of drugs and/or drug metabolites in biological matrices. J. Chromatogr. Sci. 19:392‐434.
   Guengerich, F.P. 1994. Mechanisms of formation of DNA adducts from ethylene dihalides, vinyl halides, and arylamines. Drug Metab. Rev. 26:47‐66.
   Gupta, R.C. 1985. Enhanced sensitivity of 32P‐postlabeling analysis of aromatic carcinogen‐DNA adducts. Cancer Res. 5:5656‐5662.
   Gupta, R.C. and Earley, K. 1988. 32P‐adduct assay: Comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis 9:1687‐1693.
   Halliwell, B. and Gutheridge, J. 1989. Free radicals, aging, and disease. In Free Radicals in Biology and Medicine (B. Halliwell and J. Gutheridge, eds.) pp. 416‐508. Claredon Press, Oxford.
   Ham, A.J.L., Ranasinghe, A., Koc, H., and Swenberg, J.A. 2000. 4‐Hydroxy‐2‐nonenal and ethyl linoleate form N2,3‐ethenoguanined under peroxidizing conditions. Chem. Res. Toxicol. 13:1243‐1250.
   Ham, A.J.L., Ranasinghe, A., Morinello, E.J., Nakamura, J., Upton, P.B., Johnson, F., and Swenberg, J.A. 1999. Immunoaffinity/gas chromatography/high resolution mass spectrometry method for the detection of N2,3‐ethenoguanine. Chem. Res. Toxicol. 12:1240‐1246.
   Harman, D. 1981. The aging process. Proc. Natl. Acad. Sci. U.S.A. 78:7124‐7128.
   Hermanson, G.T. (ed.). 1996. Homobifunctional cross‐linkers. In Bioconjugate Techniques. pp. 187‐227. Academic Press, San Diego.
   Kasai, H. 1997. Analysis of a form of oxidative DNA damage, 8‐hydroxy‐2′‐deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat. Res. 387:147‐163.
   Kissinger, P. 1977. Amperometric and coulometric detectors for high‐performance liquid chromatography. Anal. Chem. 49:447A‐456A.
   Koc, H., Tretyakova, N.Y., Walker, V., Henderson, R., Swenberg, J.A. 1999. Molecular dosimetry of N‐7 guanine adduct formation in mice and rats exposed to 1,3‐butadiene. Chem. Res. Toxicol. 12:566‐574.
   Krien, P.M., Margou, V., and Kermici, M. 1992. Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. J. Chromatog. 576:255‐261.
   Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., and Nishimura, S. 1987. Misreading of DNA templates containing 8‐hydroxyguanosine at the modified base and at adjacent residues. Nature 327:77‐79.
   La, D.K. and Swenberg, J.A. 1996. DNA adducts: Biological markers of exposure and potential applications to risk assessment. Mutat. Res. 365:129‐146.
   Langouët, S., Müller, M., and Guengerich, F.P. 1997. Misincorporation of dNTPs opposite 1,N2‐ethenoguanine and 5,6,7,9‐tetrahydro‐7‐hydroxy‐9‐oxoimidazo[1,2‐a]purine in oligonucleotides by Escherichia coli polymerases I exo‐ and II exo‐, T7 polymerase exo‐, human immunodeficiency virus‐1 reverse transcriptase, and rat polymerase b. Biochemistry 36:6069‐6079.
   Langouët, S., Mican, A.N., Müller, M., Fink, S.P., Marnett, L.J., Muhle, S.A., and Guengerich, F.P. 1998. Misincorporation of nucleotides opposite five‐membered exocyclic ring guanine derivatives by Escherichia coli polymerases in vitro and in vivo: 1,N2‐ethenoguanine, 5,6,7,9‐tetrahydro‐9‐oxoimidazo[1, 2‐a]purine, and 5,6,7,9‐tetrahydro‐7‐hydroxy‐9‐oxoimidazo[1, 2‐a]polymerases in vitro and in vivo: 1,N2‐ethenoguanine, 5,6,7,9‐tetrahydro‐9‐oxoimidazo[1, 2‐a]purine, and 5,6,7,9‐tetrahydro‐7‐hydroxy‐9‐oxoimidazo[1, 2‐a]. Biochemistry 37:5184‐5193.
   Lawley, P.D. 1976. Methylation of DNA by carcinogens: Some applications of chemical analytical methods. IARC Sci. Publ. 12:181‐210.
   Leung, P.Y. and Tsao, C.S. 1992. Preparation of an optimum mobile phase for the simultaneous determination of neurochemicals in mouse brain tissues by high‐performance liquid chromatography with electrochemical detection. J. Chromatog. 576:245‐254.
   Marion, M.J., Froment, O., and Trepo, C. 1991. Activation of ki‐ras gene by point mutation in human liver angiosarcoma associated with vinyl chloride exposure. Mol. Carcinogen. 4:450‐454.
   Möller, L. 1993. Optimization of an HPLC method for analyses of 32P‐postlabeled DNA adducts. Carcinogenesis 14:1343‐1348.
   Möller, L., Hofer, T., and Zeisig, M. 1998. Methodological considerations and factors affecting 8‐hydroxy‐2′‐deoxyguanosine analysis. Free Rad. Res. 29:511‐524.
   Mroczkowska, M.M. and Kusmierek, J.T. 1991. Miscoding potential of N2,3‐ethenoguanine studied in an Escherichia coli DNA‐dependent RNA polymerasein vitro system and possible role of this adduct in vinyl chloride‐induced mutagenesis. Mutagenesis. 6:385‐390.
   Nakamura, J., La, D.K., and Swenberg, J.A. 2000. 5′‐Nicked apurinic/apyrimidinic sites are resistant to β‐elimination by β‐polymerase and are persistent in human cultured cells after oxidative stress. J. Biol. Chem. 275:5323‐5328.
   Park, J.‐W. and Ames, B.N. 1988a. 7‐Methylguanine adducts in DNA are normally present at high levels and increase on aging: Analysis by HPLC with electrochemical detection. Proc. Natl. Acad. Sci. U.S.A. 85:7467‐7470.
   Park, J.‐W. and Ames, B.N. 1988b. Correction. Proc. Natl. Acad. Sci. U.S.A. 85:9508.
   Park, J.‐W., Cundy, K., and Ames, B.N. 1989. Detection of DNA adducts by high‐performance liquid chromatography with electrochemical detection. Carcinogenesis 10:827‐832.
   Ranasinghe, A., Scheller, N.A., Wu, K.Y., Upton, P.B., and Swenberg, J.A. 1998. Application of gas chromatography/electron capture negative chemical ionization high‐resolution mass spectrometry for analysis of DNA and protein adducts. Chem. Res. Toxicol. 11:520‐526.
   Randerath, K. and Randerath, E. 1994. 32P‐Postlabeling methods for DNA adduct detection: Overview and critical evaluation. Drug Metab. Rev. 26:67‐85.
   Randerath, K., Reddy, M.V., and Gupta, R.C. 1981. 32P‐Postlabeling test for DNA damage. Proc. Natl. Acad. Sci. U.S.A. 78:6126‐6129.
   Reddy, M.V. and Randerath, K. 1986. Nuclease P1‐mediated enhancement of sensitivity of 32P‐postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7:1543‐1551.
   Richter, C., Park, J.‐W., and Ames, B.N. 1988. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U.S.A. 85:6465‐6467.
   Schneider, C., Newman, R.A., Sutherland, D.R., Asser, U., and Greaves, M.F. 1982. A one‐step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem. 257:10766‐10769.
   Singer, B., Spengler, S.J., Chavez, F., and Kusmierek, J.T. 1987. The vinyl chloride‐derived nucleoside, N2,3‐ethenoguanosine, is a highly efficient mutagen in transcription. Carcinogenesis. 8:745‐747.
   Singer, B., Kusmierek, J.T., Folkman, W., Chavez, F., and Dosanjh, M.K. 1991. Evidence for the mutagenic potential of the vinyl chloride‐induced adduct, N2,3‐etheno‐deoxyguanosine, using a site‐directed kinetic assay. Carcinogenesis 12:745‐747.
   Sodum, R.S. and Chung, F.L. 1988. 1,N2‐ethenodeoxyguanosine as a potential marker for DNA adduct formation by trans‐4‐hydroxy‐2‐nonenal. Cancer Res. 48:320‐323.
   Sodum, R.S. and Chung, F.L. 1989. Structural characterization of adducts formed in the reaction of 2,3‐epoxy‐4‐hydroxynonanal with deoxyguanosine. Chem. Res. Toxicol. 2:23‐28.
   Sodum, R.S. and Chung, F.L. 1991. Stereoselective formation of in vitro nucleic acid adducts by 2,3‐epoxy‐4‐hydroxynonanal. Cancer Res. 51:137‐143.
   Swenberg, J.A., Bogdanffy, M.S., Hani, A., Holt, S., Kim, A., Morinello, E.J., Ranasinghe, A., Scheller, N., and Upton, P.B. 2000. Formation and repair of DNA adducts in vinyl chloride‐ and vinyl fluoride‐induced carcinogenesis. In Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. IARC Scientific Publications No. 150 (B. Singer and H. Bartsch, eds.) pp. 29‐43. International Agency for Research on Cancer, Lyons, France.
   Swenberg, J.A., Richardson, F.C., Boucheron, J.A., and Dyroff, M.C. 1985. Relationships between DNA adduct formation and carcinogenesis. Environ. Health Perspec. 62:177‐183.
   Tretyakova, N.Y., Chiang, S.Y., Walker, V.E., and Swenberg, J.A. 1998. Quantitative analysis of 1,3‐butadiene‐induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 33:363‐376.
   van Delft, J.H.M., Steenwinkel, M.‐J.S.T., de Groot, A.J.L., van Zeeland, A.A., Eberle‐Adamkiewicz, G., Rajewsky, M.F., Thomale, J., and Baan, R.A. 1997. Determination of N7‐ and O6‐methylguanine in rat liver DNA and oral exposure to hydrazine by use of immunochemical and electrochemical detection methods. Fund. Appl. Toxicol. 35:131‐137.
   Walker, V.E., Wu, K.‐Y., Upton, P.B., Ranasinghe, A., Scheller, N., Cho, M.‐H., Vergnes, J.S., Skopek, T.R., and Swenberg, J.A. 2000. Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide. Carcinogenesis 21:1661‐1669.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library