Methods for Measuring DNA Adducts and Abasic Sites I: Isolation, Purification, and Analysis of DNA Adducts in Intact DNA

James A. Swenberg1, Amy‐Joan L. Ham1, Kevin S. McDorman1, Eric J. Morinello1, Jun Nakamura1, Robert Schoonhoven1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.8
DOI:  10.1002/0471140856.tx0308s12
Online Posting Date:  August, 2002
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Abstract

The major event involved in the formation of mutations and the initiation and progression of cancer is the induction of DNA damage by reactive intermediates arising from exposure to endogenous and exogenous chemicals. Many electrophilic metabolites of chemicals covalently bind to the bases of DNA causing specific DNA adducts. This unit includes protocols for preparing samples of intact DNA and adduct analysis to quantify the number of adducts that can potentially cause mutagenic or carcinogenic damage.

     
 
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Table of Contents

  • DNA Isolation Methods
  • Basic Protocol 1: Low‐Temperature/Antioxidation DNA Isolation
  • Alternate Protocol 1: Room Temperature/Antioxidation DNA Isolation
  • Support Protocol 1: Spectrophotometric Quantitation of DNA
  • Basic Protocol 2: Measurement of Apurinic/Apyrimidinic Sites
  • Basic Protocol 3: Measurement of DNA Adducts by Immuno‐Slot Blot (ISB)
  • Basic Protocol 4: Immunohistochemical Demonstration of DNA Adducts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Low‐Temperature/Antioxidation DNA Isolation

  Materials
  • 10 mg/ml proteinase K, nucleic acid purification grade (PE Biosystems): store powder up to 1 year at 4°C and solution up to 1 year at −20°C
  • 1× lysis buffer/20 mM 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (lysis buffer/TEMPO; see recipe)
  • 1× phosphate buffered saline without Ca2+ or Mg2+ (CMF‐PBS; see recipe)/20 mM TEMPO
  • 20 mg/ml butylated hydroxytoluene (BHT; Sigma‐Aldrich) in isopropanol: store up to 1 month at −20°C
  • Tissue
  • 70% phenol/chloroform/water (Applied Biosystems): store up to 6 months at 4°C
  • Sevag mixture (Applied Biosystems): store up to 6 months in a brown bottle at room temperature
  • 2 M and 1 mM TEMPO (2,2,6,6‐tetramethyl‐1‐piperidinyloxy; Sigma‐Aldrich)
  • 4 M NaCl: store up to 4 weeks at room temperature
  • 100% and 70% ethanol, −20°C
  • 30 mg/ml (∼3000 Kunitz units/ml) ribonuclease A (Sigma‐Aldrich)
  • 5000 U/ml ribonuclease T 1 (Roche or Sigma‐Aldrich): boil 15 min to inactivate DNases and store up to 6 months at 4°C
  • 15‐ and 50‐ml polypropylene centrifuge tubes
  • Tissue homogenizer with a glass tube and loose‐fitting Teflon pestle with >0.1 to 0.15 mm tolerance
  • 10‐ml pipets
  • Additional reagents and equipment for determining DNA concentration (see protocol 3)
CAUTION: TEMPO is a hazardous chemical that is corrosive, combustible, and readily absorbed through the skin. Use only in a chemical fume hood and wear suitable protection. Refer to the material safety data sheet (MSDS) for more information.

Alternate Protocol 1: Room Temperature/Antioxidation DNA Isolation

  • Tissue or pelleted sample
  • 1× phosphate‐buffered saline without Ca2+ or Mg2+(CMF‐PBS; see recipe)
  • 1× lysis buffer/BHT: add 1 ml of 20 mg/ml BHT (Sigma‐Aldrich) in isopropanol per 450‐ml bottle of 1× lysis buffer (Applied Biosystems)
  • RNase solution (see recipe)
  • 70% phenol/water/chloroform containing BHT: add 1 ml of 20 mg/ml BHT in isopropanol per 450‐ml bottle of 70% phenol/water/chloroform (Applied Biosystems)
  • Chloroform/BHT (see recipe)
  • 3 M sodium acetate trihydrate (Mallinckrodt; appendix 3A)
  • 95% ethanol, −20°C
  • Isopropanol (Mallinckrodt)
  • Alumina (Sigma‐Aldrich): store up to 6 months in a 60°C oven
  • Wheaton overhead mixer
  • Gel‐loading tips
  • 3‐ml syringes and 22‐G needles
CAUTION: Phenol is a caustic chemical and contact with skin should be avoided. Perform this step under a fume hood using only glass pipets as chloroform/phenol will melt some plastic pipets. Insert only the tip of the pipet into solution as the numbers and lines may be removed by the organic solvents.

Support Protocol 1: Spectrophotometric Quantitation of DNA

  Materials
  • DNA samples (see protocol 1 or protocol 2)
  • Spectrophotometer with reference and sample slots
  • Quartz cuvettes with 1 cm path length

Basic Protocol 2: Measurement of Apurinic/Apyrimidinic Sites

  Materials
  • One primary (e.g., Dojindo) and three secondary (e.g., Dojindo or equivalent) internal DNA standards (100 and 30 AP sites/1 × 106 nucleotides, respectively)
  • Unknown DNA samples
  • 10× PBS, pH 7.4 (see recipe)
  • 10 mM aldehyde‐reactive probe (ARP; Dojindo Molecular Technologies): store up to 1 year at −80°C
  • 100% and 70% ethanol, −20°C
  • 1× TE buffer, pH 7.5 (see recipe for 10×)
  • Calf thymus DNA (Sigma‐Aldrich)
  • 1 and 2 M ammonium acetate
  • 5× SSC (see recipe)
  • Hybridization mix (see recipe)
  • Streptavidin‐conjugated horseradish peroxidase (HRP; BioGenex Laboratories)
  • Washing buffer (see recipe)
  • ECL reagent (Amersham)
  • Minifold II dot/slot blotting manifold (Schleicher & Schuell)
  • Blotting paper (Schleicher & Schuell)
  • Nitrocellulose (NC) membrane—e.g., Hybond C‐Super (Amersham), BA 85 (Schleicher & Schuell)
  • 37°C environmental shaker
  • 80°C vacuum oven
  • Bottle rotator
  • Scanning densitometer
  • Temperature‐controlling device (e.g., thermocycler, water bath, etc.)
  • Additional reagents and equipment for measurement of DNA concentration (see protocol 3)

Basic Protocol 3: Measurement of DNA Adducts by Immuno‐Slot Blot (ISB)

  Materials
  • Internal standard containing 30 DNA lesions/1 × 106 nucleotides
  • Unknown DNA samples
  • 1× TE buffer, pH 7.5 (see recipe for 10×)
  • Calf thymus DNA (Sigma‐Aldrich)
  • 1 and 2 M ammonium acetate
  • 5× SSC (see recipe)
  • Antibody blocking buffer (see recipe)
  • Primary antibody against specific DNA lesions (e.g., R & D Systems)
  • Washing buffer (see recipe)
  • Hybridization mix (see recipe)
  • Biotinylated secondary antibody (BioGenex Laboratories)
  • Streptavidin‐conjugated HRP (BioGenex Laboratories)
  • ECL reagent (Amersham western kit)
  • 37°C environmental shaker
  • 80°C vacuum oven
  • Bottle rotator
  • Additional reagents and equipment for set up of Minifold dot/slot blotting manifold (see protocol 4, step )

Basic Protocol 4: Immunohistochemical Demonstration of DNA Adducts

  Materials
  • Slides with tissue sections, treated and control
  • Xylene
  • 100%, 95%, and 70% ethanol
  • 1× PBS (see recipe for 10×)/1% (v/v) Tween 20
  • DAKO Envision Kit for HRP (Dako):
  •  Blocking reagent: 0.3% H 2O 2 in methanol
  •  Polymer‐labeled secondary antibody
  •  DAB
  •  Substrate buffer
  • Primary antibody
  • Quick DAB enhancer solution (Innovex Biosciences)
  • Aqua Hematoxyin (Innovex Biosciences)
  • Permount (Fisher Scientific)
NOTE: Other manufacturers' reagents can be substituted; however, incubation times in the following procedure would have to be changed according to their directions.NOTE: All of the following steps are performed at room temperature.
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Figures

Videos

Literature Cited

Literature Cited
   Brent, T.P., Teebor, G.W., and Duker, N.J. 1978. Lesions in alkylated DNA determined by susceptibility to alkali, apurinic endonuclease or N‐glycosylase In DNA Repair Mechanisms (P.C. Hanawalt, E.C. Friedberg and C.F. Fox eds.) pp. 19‐22. Academic Press, New York.
   Chung, F.L., Chen, H.J., and Nath, R.G. 1996. Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17:2105‐2111.
   Dragan, Y.P., Hully, J.R., Nakamura, J., Mass, M.J., Swenberg, J.A., and Pitot, H.C. 1994. Biochemical events during initiation of rat hepatocarcinogenesis.Carcinogenesis 15:1451‐1458.
   Farmer, P.B. and Shuker, D.E. 1999. What is the significance of increases in background levels of carcinogen‐derived protein and DNA adducts? Some considerations for incremental risk assessment. Mutat. Res. 424:275‐286.
   Greferath, R. and Nehls, P. 1997. Monoclonal antibodies to thymidine glycol generated by different immunization techniques. Hybridoma 16:189‐193.
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   Loeb, L.A. and Preston, B.D. 1986. Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet. 20:201‐230.
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   Maulik, G., Botchway, S., Chakrabarti, S., Tetradis, S., Price, B., and Makrigiorgos, G.M. 1999. Novel non‐isotopic detection of MutY enzyme‐recognized mismatches in DNA via ultrasensitive detection of aldehydes. Nucl. Acids Res. 27:1316‐1322.
   Nakamura, J. and Swenberg, J.A. 1999. Endogenous apurinic/apyrimidinic sites in genomic DNA of mammalian tissues. Cancer Res. 59:2522‐2526.
   Nakamura, J., Walker, V.E., Upton, P.B., Chiang, S.‐Y., Kow, Y.W., and Swenberg, J.A. 1998. Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res. 58:222‐225.
   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.
   Nehls, P., Adamkiewicz, J., and Rajewsky, M.F. 1984. Immuno‐slot‐blot: A highly sensitive immunoassay for the quantitation of carcinogen‐modified nucleosides in DNA. J. Cancer Res. Clin.Oncol. 108:23‐29.
   Phillips, J.C., Davies, S., and Lake, B.G. 1999. Dose‐response relationships for hepatic aflatoxin B1‐DNA adduct formation in the rat in vivo and in vitro: The use of immunoslot blotting for adduct quantitation. Teratogen. Carcinog. Mutagen. 19:157‐170.
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