U.S. Dept Commerce/NOAA/NMFS/NWFSC/Publications NOAA-NWFSC Tech Memo-14: 32P-Postlabeling Protocols for Assaying Levels of Hydrophobic DNA Adducts in Fish NOAA Technical Memorandum NMFS-NWFSC-1432P-Postlabeling Protocols for Assaying Levels of       Hydrophobic DNA Adducts in FishWilliam L. Reichert and Barbara FrenchNational Marine       Fisheries ServiceNorthwest Fisheries Science CenterCoast Zone and       Estuarine Studies Division2725 Montlake Blvd. E.Seattle WA       98112-2097       
June 1994
U.S. DEPARTMENT OF COMMERCERonald H. Brown,       Secretary       
National Oceanic and Atmospheric AdministrationD.       James Baker, Administrator

National Marine Fisheries ServiceRolland A.       Schmitten, Assistant Administrator for Fisheries 
PREFACE
The Environmental Conservation (EC) Division of the Northwest Fisheries Science Center is evaluating biochemical parameters for use as markers of chemical contaminant exposure and early physiological effects induced by such exposure. A very promising approach is the use of the 32P-postlabeling assay for determining levels of hydrophobic aromatic compounds bound to DNA (DNA adducts) in marine organisms. Recent publications from the EC Division have shown that the 32P-postlabeling method can be used to detect and measure the levels of DNA modified by environmental genotoxic compounds in feral fish. These studies have shown that 1) the levels of hepatic DNA adducts in wild fish positively correlate with the concentrations of polycyclic aromatic hydrocarbons (PAHs) present in marine sediments, and 2) that a strong positive correlation is observed between sediment concentrations of PAHs and the prevalence of neoplastic lesions in liver of marine flatfish. In addition, laboratory studies with model PAHs and sediment extracts have shown that PAH-DNA adducts formed are persistent and have chromatographic characteristics similar to adducts detected in wild fish. These findings suggest that the levels of hepatic DNA adducts found in fish tissues may function as molecular dosimeters of exposure to potentially genotoxic environmental contaminants, such as carcinogenic PAHs. The 32P-postlabeling assay is currently being used as a marker of exposure to potentially genotoxic contaminants in environmental monitoring studies, such as the National Benthic Surveillance Project of NOAA's National Status and Trends (NS&T) Program and in the Bioeffects Surveys of NOAA's Coastal Ocean Program. This NOAA technical memorandum describes in detail the 32P- postlabeling method and its application to marine organisms.

CONTENTS
- PREFACE 
- INTRODUCTION    
- LABORATORY   SETUP 
- Facilities    
- Equipment    
- CHEMICALS    
- RADIATION   SAFETY  
- Equipment   for Handling 32P  
- Radioactive   Waste and Disposal  
- POLYETHYLENEIMINE-MODIFIED   CELLULOSE SHEETS     - Commercial     PEI-Cellulose Sheets  
  - Preparation     of PEI-Cellulose Sheets  
  - Preparation     of 0.5% PEI Solution  
  - Preparation     of Vinyl Backing for Chromatography Sheets  
  - Preparation     and Pouring of PEI-Cellulose Slurry  
  - Cutting,     Washing and Storage of PEI-Cellulose Sheets 
- SYNTHESIS   OF [gamma-32P]ATP     - Preparation     and Storage of [gamma-32P]ATP Synthesis Premix  
  - [gamma-32P]ATP     Synthesis Procedure  
  - Determination     of [gamma-32P]ATP Specific Activity  
  - Reagents      
  - Assay     Protocol  
  - Sample     Calculation for Determining Specific Activity 
- DNA   ISOLATION  
- Manual   Procedure  
- Automated   DNA Extraction Procedure  
- Reagents   for DNA Isolation  
- ENZYMATIC   HYDROLYSIS OF DNA  
- Procedure   for Digestion of DNA Samples  
- DNA-ADDUCT   ENHANCEMENT PROCEDURES     - Butanol     Adduct Enhancement  
  - Procedure      
  - Nuclease     P1 Enhancement  
  - Procedure 
- 32P-POSTLABELING   OF DNA ADDUCTS AND BASES  
- Procedure   for 32P-Postlabeling of DNA Adducts  
- Procedure   for 32P-Labeling of Bases  
- CHROMATOGRAPHY    
- Preparation   of Chromatography Solvents  
- Chromatography   of Xenobiotic DNA Adducts  
- Alternative   D4 Solvent Systems  
- IMAGING   AND QUANTITATION OF RADIOACTIVITY ON CHROMATOGRAMS     - Autoradiography      
  - Procedure      
  - Photography     of Autoradiograms  
  - Quantitation     of Radioactivity by the "Cut and Count" Method  
  - Liquid     Scintillation Spectrometry  
  - Cerenkov     Counting  
  - Storage     Phosphor Imaging  
  - Scanning     the Chromatograms  
  - Data     Storage and Computer Requirements  
  - Processing     of Image Analysis Data  
  - Calculations      
  - Sample     calculations 
- QUALITY   ASSURANCE/QUALITY CONTROL  
- ACKNOWLEDGMENTS    
- CITATIONS    
- GLOSSARY   OF ABBREVIATIONS  
- FIGURES    
- TABLE   
INTRODUCTION
The 32P-postlabeling (PPL) method for detection of DNA adducts was developed in Dr. Kurt Randerath's laboratory in the early 1980s (Gupta et al. 1982) and has evolved substantially since then. Currently the 32P-postlabeling technique is the most sensitive method for the detection of a wide range of compounds bound to DNA. For hydrophobic, aromatic DNA adducts, such as polycyclic aromatic hydrocarbon PAH-DNA adducts, this method can detect 1 adduct in 109-1010 bases by selective removal of unmodified nucleotides by enzymatic methods (Reddy and Randerath 1986) or by partitioning the DNA adducts into n-butanol (Gupta 1985). The DNA adducts are then phosphorylated via enzyme catalyzed transfer of 32P-phosphate from [gamma-32P]ATP to the deoxyribose of the adduct. Further, the nonspecific nature of the 32P-postlabeling assay allows for the detection of a wide range of bulky, hydrophobic compounds bound to DNA. This attribute coupled with the high sensitivity of the assay has led to the broad use of the 32P-postlabeling assay in studies with mammals and fish for assessing exposure to environmental genotoxins (Dunn et al. 1987, Varanasi et al. 1989a, Liu et al. 1991, Poginsky et al. 1990, Ray et al.1991, Stein et al. 1992) and to specific genotoxic compounds, such as benzo[a]pyrene (BaP) and 7H-dibenzo[c,g]carbazole (DBC) (Randerath et al. 1984, Schurdak et al. 1987, Varanasi et al. 1989b, Sikka et al. 1991, Stein et al. 1993).

In 1987, we initiated studies using the 32P-postlabeling assay for evaluating exposure of marine fish to environmental carcinogens in order to assess relationships between carcinogen exposure and the development of hepatic neoplasia, which are observed in several benthic marine species from contaminated coastal areas of the United States (Johnson et al. 1993, Myers et al. 1993). To date we have analyzed hepatic DNA samples from more than 20 species of marine fish sampled from numerous reference and contaminated sites on the U.S. coast as part of ongoing NOAA Programs, such as the National Benthic Surveillance Project of the National Status and Trends Program and the Bioeffects Surveys of the Coastal Ocean Program. In addition, we have conducted laboratory studies to determine the kinetics of formation and removal of DNA adducts in fish (Stein et al. 1993) and to determine the identity of the adducts observed (Varanasi et al. 1989a). Moreover, these studies led to modifications in the 32P-postlabeling assay to improve the application of this technique to feral fish.

The detection of DNA adducts by 32P-postlabeling is a multistep sequence (Fig. 1) involving a series of biochemical reactions. Initially, DNA is hydrolyzed enzymatically to 3'-monophosphates. The digest is then enriched in xenobiotic-modified mononucleotides by the selective removal of normal nucleotides. Following the enrichment step the adducted DNA is enzymatically labeled at the 5'-hydroxyl position with [32P]phosphate to form [5'-32P]deoxyribonucleoside 3',5'-bisphosphates. Separation of the 32P-labeled adducts is usually accomplished by two-dimensional, thin-layer chromatography (TLC) on polyethyleneimine (PEI)-modified cellulose sheets. Autoradiography or storage phosphor imaging (Reichert et al. 1992) is then used to locate the radiolabeled adducts on the chromatogram. The radioactivity on the chromatograms can then be quantitated by liquid scintillation spectrometry or storage phosphor imaging.

The accurate quantitation of individual adducts is dependent on the specificity and efficiency of the enzymes used, which can vary substantially for each type of adduct. Optimization of each enzymatic step for specific adducts is required to increase the accuracy of measuring individual adduct levels (Watson 1987, Gorelick and Wogan 1989). Currently, the method can be considered only semiquantitative in organisms, such as feral fish, exposed to complex mixtures of environmental contaminants that have not been fully characterized. However, because mixtures of chemical contaminants are poorly characterized, the ability of the 32P-postlabeling assay to detect adducts of unknown structure is a key attribute.

In this NOAA technical memorandum, the procedures and equipment we currently use in our laboratories are described. Each section discusses the function and biochemical basis for a particular step and the methodology and the procedural pitfalls. The samples we typically analyze are hepatic tissues from fish; however, the method is applicable to any tissue from which DNA can be extracted. A glossary of abbreviations is provided at the end of the text.
LABORATORY SETUP 
Facilities

A dedicated laboratory is used for the 32P-postlabeling analysis. This laboratory contains a fume hood, sink, -20°C freezer, -80°C freezer and a refrigerator. The fluorescent ceiling lights all have 400 nm cutoff to reduce photodegradation of samples.
Equipment Centrifugal Vacuum EvaporatorSavant Speed-Vac SVC100H (footnote 1) (Savant Instruments, Farmingdale, NY 11735) equipped with a glass cover (unaffected by organic solvents) for removal of solvents from samples held in the microcentrifuge tubes. A liquid nitrogen trap is placed between the Speed-Vac and the vacuum pump (Duoseal Vacuum Pump) to trap solvents coming off the samples. 
Refrigerated Microcentrifuge Eppendorf Model 5402 refrigerated microcentrifuge is used in postlabeling and DNA extraction procedures.

Tabletop Centrifuge A Sorvall T6000B (DuPont) is used for centrifuging the Plexiglas carousels holding the radioactive samples during postlabeling.

Spreader for Making Thin-Layer Chromatographic (TLC) SheetsA DeSaga TLC spreading device (Desaga 120305 or Whatman 49961-102).

Blender A variable speed/pulse kitchen blender to mix PEI/cellulose solutions.

Radiation Monitor A pancake-type radiation monitor (Technical Associates PUG 1 AB, Canoga Park, CA) is used for the detection of 32P contamination in the laboratory (see section on Radiation Safety).

Autoradiography Equipment Any 14" x 17" metal autoradiography cassette will work. The cassettes are lined on one inner side with DuPont Cronex Lightning-Plus intensifying screens.

Darkroom The darkroom is equipped with a safelight, a sink, developer, fixer, and waterbath trays for developing the 14" x 17" negatives.

Tissue HomogenizersA Polytron PT 1200C with a 5 mm generator (Brinkmann Instruments, Westbury, NY) or a glass Dounce homogenizer (5 to 10 mL size) is used for tissue homogenization.

Waterbaths Any standard tabletop waterbath (20-60°C range) can be used for incubations and enzyme hydrolyses. A separate waterbath is used for radiolabeled samples.

Thermomixer Eppendorf Thermomixer 5436 from Brinkmann Instruments (Westbury, NY).

Plexiglas Carousels Plexiglas carousels are used for holding the microcentrifuge tubes containing radioactive samples during incubations. Please see Reddy and Blackburn (1990) for design specifications.

Thin-Layer Chromatography (TLC) Tanks Standard glass TLC tanks (inside dimensions 275 mm x 275 mm x 75 mm) can be used; however, if large numbers of samples are processed then the Plexiglas multisheet holders (see Reddy and Blackburn 1990 for specifications) are preferable.

SpectrophotometerShimadzu UV/VIS Model 2100. Quartz cuvettes with a 1 cm path length and 4 mm width are used for measuring DNA absorbances.

Analytical Balance Mettler AC100 from Mettler Instrument Co., Hightstown, NJ.

Microcentrifuge Tubes Any high quality microcentrifuge tube (0.5, 1.5 and 1.9 mL) will work; however, all tubes should be methanol rinsed. Occasionally a residue is present on the tubes that can inhibit enzyme activity.

Freezers A -70°C (or colder) freezer must be used for the storage of all tissues and purified DNA. A -20°C freezer may be used for overnight storage of samples but never for extended periods.

Liquid Nitrogen DewarsUsed for quick freezing of tissue samples in liquid nitrogen.

Phosphorescent Ink Pens A phosphorescent ink pen (NEN, DuPont) is used to mark the chromatograms so that the autoradiograms can be aligned with the chromatograms. The radioactive areas on the chromatograms can then be marked for excision.

Pipettors We use both the Eppendorf and Gilson adjustable micropipettors in sizes of 10, 20, 100, 200 and 1000 μL. One set of pipettors is used for radioactive samples and a separate set is used for nonradioactive samples.

Shielding See section on Radiation Safety.

Liquid Scintillation Counter We use a Packard Instrument Company Model 1900 TR liquid scintillation counter (Downers Grove, IL).

Storage Phosphor Imaging System Storage phosphor imaging screens (14" x 17", MD23-614) are scanned on a Model 425E PhosphoImager (Molecular Dynamics, Sunnyvale, CA). This system comes with 486-33 mHz computer for data processing.

Computer A computer is necessary if you plan to use storage phosphor imaging technology to image and quantify the chromatograms generated by 32P-postlabeling. The files generated are large (up to 41 MB) and it is recommended that a 386 SX or preferably a 486 PC with at least a 120 MB hard disk and 8 MB or more of RAM be used for data processing. A high quality monitor is essential for processing the images (such as Sony 1304 monitor). Currently, we are using a 486-33 MHz with a 330 MB hard disk, 48 MB of RAM, and a video accelerator card. With this configuration we are able to process and quantitate the data in a virtual RAM drive, which is three to six times faster than working off the hard disk.

pH Meter and Electrodes Any quality pH meter is acceptable. For preparation of Tris buffers it is recommended that a calomel rather than a silver chloride pH electrode be used.

Gloves For all laboratory work, disposable latex gloves are used (see Radiation Safety section.).

Hot-Air Blow Dryers Any quality hair dryer will work for drying PEI-cellulose TLC sheets between chromatography steps.

Stirring Motor A variable speed stirring motor is used for agitating the water in the rinse tanks used to remove salts from the chromatography sheets.

Automated DNA Extractor We use Applied Biosystems Inc. (Foster City, CA) Genepure 341 Nucleic Acid Purification System.
CHEMICALS
Carrier-free (32P)phosphate (NEX-053) was obtained from New England Nuclear Research Products (E. I. DuPont, Wilmington, DE) and carrier-free [gamma-32P]ATP (5-6x103 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). The following were obtained from Sigma Chemical (St. Louis, MO): Bicine (B-3876), Tris (T-1503), CHES buffer (C-2885), urea (U-1250), spermidine (S-2626), dithiothreitol (D-0632), L-glycerol-3-phosphate (G-7886), adenosine 5'-diphosphate (A-6521), adenosine 5'-triphosphate (A-6144), 2'-deoxyadenosine 3'-monophosphate (D-3014), sodium pyruvate (P-2256), lithium chloride (L-0505), proteinase K (P-0390), nuclease P1 (N-8630), micrococcal endonuclease (N-3755), spleen phosphodiesterase (P-6897), apyrase (A-6132), RNase A (R-4875), RNase T1 (R-8251), RNase T2 (R-3751), polyethyleneimine in 50% aqueous solution (P-3143), and alpha-amylase (A-6255). Cloned T4 polynucleotide kinase (70031) was purchased from United States Biochemical (Cleveland, OH). Cellulose powder (MN-301 is manufactured by Macherey Nagel in Germany, Brinkman 6610100-8) was obtained from Brinkmann, Westbury, NY. Molecular biology grade phenol was purchased from Boehringer Mannheim (Indianapolis, IN). Chemicals used on the automated DNA extractor such as phenol/chloroform/water, lysis buffer, chloroform and 5 M sodium acetate were purchased from Applied Biosystems Inc. (Foster City, CA). All other chemicals used were reagent grade or better.

Enzymes used for the synthesis of [gamma-32P]ATP were as follows: glycerol-3-phosphate dehydrogenase (127124), triosephosphate isomerase (109754), glyceraldehyde-3-P-dehydrogenase (105686), lactate dehydrogenase (127230), 3-phosphoglycerate kinase (108430), and ß-NAD (127302); these were purchased from Boehringer Mannheim (Indianapolis, IN).

For preparation of assay reagents, we use either double distilled, deionized water or high performance liquid chromatography (HPLC) grade water. For preparation of reagents, please see appropriate sections.
RADIATION SAFETY
The 32P-postlabeling assay uses large amounts of 32P, which is an energetic beta emitter (1.7 MeV). Therefore, any person using this isotope must receive detailed instruction before handling 32P and must be frequently monitored for exposure to 32P. Since an inexperienced person may not realize how easily radioactivity is unintentionally spread, pretraining with a fluorescent solution may be necessary. A new person should perform laboratory operations using a fluorescent solution (i.e. fluorescein, quinine sulfate) and then turn off the overhead lights and use a black light to reveal handling errors that would have resulted in unwanted spreading of radioactivity.

Important points to help minimize and monitor 32P exposure:
- All employees who handle 32P must wear a whole body film badge   and a thermiluminescent type finger ring dosimeter on the inside of each hand   where there is the highest potential for radiation exposure. Rings and badges   are monitored monthly.   
- All laboratory operations are planned to minimize the time spent handling   radioactivity.   
- Double latex gloves are worn while handling 32P and they are   frequently checked for radioactivity by passing them under a pancake-type   radiation monitor. If gloves are constantly needing to be changed because of   32P contamination, sample handling procedures must be reviewed and   revised.   
- Workers wear labcoats and use disposable sleeves.   
- Laboratory working surfaces are checked frequently with the radiation   monitor when handling 32P. The monitor probe should be covered with   a thin vinyl wrap to prevent contamination of the probe.   
- After completion of work with radioactivity, the workers are to check   themselves and their equipment with the radiation monitor. If any   radioactivity is detected then they are to wash themselves and/or the   equipment until free of radioactivity.   
- A senior staff member experienced in handling radioactivity routinely   checks the above points to ensure that correct procedures are followed. 
Equipment for Handling 32P
All 32P is handled behind 10 to 13-mm Plexiglas shielding. In addition, samples are kept in Plexiglas containers that are at least 13-mm thick. Most of the Plexiglas equipment we use (e.g., carousels for holding the microcentrifuge tubes, shields for the pipettors, and racks for holding the chromatograms during chromatography and drying) is similar to that described by Reddy and Blackburn (1990). This equipment increases sample handling capacity, while substantially lowering radiation exposure risk.
Radioactive Waste and Disposal
Radioactive waste is temporarily stored in a remote corner of the laboratory in a 13-mm thick Plexiglas box that has a 1.5-mm thick lead foil covering (Josefsen et al. 1993). This container is emptied regularly and the 32P waste is transported to a designated storage site for radioactive materials. Radioactive waste is stored in a locked shed in a 55-gallon steel drum labeled "32P only." Once a drum is filled, it is sealed and dated. After 10 half-lives (143 days) the contents of the drum are scanned with a survey meter equipped with a pancake-type detector. If there is no reading on the survey meter then the contents of the barrel are disposed of in the regular waste.

Make sure that 3H-, 14C-labeled compounds and carcinogens are not put into the 32P waste barrel.

For additional information on the safe handling of 32P see Ballance et al. (1984) and Slobodien (1980).
POLYETHYLENEIMINE-MODIFIED CELLULOSE SHEETS
Satisfactory chromatography of hydrophobic DNA adducts can be achieved with either commercially available or laboratory-prepared polyethyleneimine-modified cellulose (PEI-cellulose) TLC sheets. We have found that the laboratory prepared PEI-cellulose TLC sheets can give sharper solvent fronts and that the spot resolution is generally superior to that obtained from commercial PEI-cellulose sheets. If you plan to do 32P-postlabeling on a large scale, it is cost effective to make your own PEI-cellulose sheets. However, laboratory prepared sheets are not as durable as the commercial sheets and must be handled with greater care.
Commercial PEI-Cellulose Sheets
The performance of commercially obtained PEI-cellulose sheets can be improved by shaking the sheets gently for 2-4 minutes in reagent grade methanol to remove impurities from the manufacturing process. After the methanol wash, the sheets are shaken in distilled water for 10 minutes. The washed sheets are air dried thoroughly and wrapped with aluminum foil or placed in a sealed container for storage; they are stable at -20°C for at least 2 months. The PEI-cellulose sheets manufactured by Macherey Nagel (available through Alltech) are satisfactory for chromatography.
Preparation of PEI-Cellulose Sheets
The procedure used for preparation of PEI-cellulose sheets is based on the method of Randerath and Randerath (1964). Currently, we use PEI in a 50% aqueous solution (P-3143) purchased from Sigma Chemical.

Note: The cellulose used is produced by Macherey Nagel and is now called MN-301. This product is more difficult to work with than its predecessor MN-300.

Preparation of 0.5% PEI Solution

For 1 liter of solution, 900 mL of deionized distilled water (ddH2O) is added to 10 g of a 50% PEI solution in a beaker. The pH is adjusted to approximately 6 with 6N HCl while constantly stirring. Initially, PEI is a clear, viscous solution at the bottom of the beaker, but as the pH is lowered the PEI will go into solution. After the PEI has dissolved and a pH of 6 has been reached, bring the solution to a final volume of 1 liter. This solution is stable for up to 4 months when stored at 0-4°C. The retentive characteristics of the PEI-modified cellulose sheets can be increased by raising the percentage of PEI present in the solution.

Preparation of Vinyl Backing for Chromatography Sheets
- Scrub the dull surface of a matte finish vinyl sheet   (8.5"x 50", 0.01" thick, Universal Plastics, Seattle, WA) with a commercial   cleaner and rinse thoroughly with soft tap water or distilled water.   
- Place the vinyl sheet on a clean glass plate of the same dimensions (the   glass plate provides a rigid backing for the vinyl strip) with the dull side   facing up. Be sure that one of the long edges of the glass plate barely   overhangs the countertop edge. This allows the TLC spreader to slide smoothly   along the vinyl sheet.   
- Dry the vinyl sheet.   
- Set the TLC spreader opening at 0.4 mm.   
- Run TLC spreader along the vinyl sheet to check for smoothness and remove   any gritty material that is detected either on top of the vinyl strip or   between the vinyl strip and the glass plate. 

Preparation and Pouring of PEI-Cellulose Slurry
- Place 34 g of MN-301 cellulose in a blender.   
- Add 145 mL of a 0.5% PEI solution.   
- Pulse the blender a few times and then "liquefy" on low for 15 seconds.   
- Pour the PEI-cellulose slurry into a vacuum flask and turn on the   aspirator. Swirl the flask vigorously while aspirating to   prevent the PEI-cellulose slurry from foaming. This step may take 10 to 15   minutes because the PEI-cellulose slurry is slow to degas. Removal of   the dissolved gases is important because bubbles will affect adherence of the   PEI-cellulose to the vinyl sheets and leave small holes in the cellulose   matrix.   
- Place the TLC spreader at one end of the vinyl sheet.   
- Pour the PEI-cellulose slurry gently into the spreader to avoid   unnecessary generation of bubbles and stir gently with glass rod to remove any   bubbles.   
- Turn the spreader handle and immediately spread the slurry in one slow   steady movement along the vinyl strip. At the end of the vinyl strip turn the   spreader handle back to the closed position. This step will require some   practice to evenly spread the slurry. The slurry layer is usually thicker at   the ends of the vinyl strip. If pinhead sized holes are present in the slurry   layer, press these dry spots with a fine pointed tool which will cause the   slurry to fill the void. Wait for approximately 3 hours before removing the   sheets from the glass plates.   
- Dry the PEI-cellulose sheets overnight. 

Cutting, Washing and Storage of PEI-Cellulose Sheets
- Cut the sheets into 20 cm widths with a paper cutter (do not use sections   that were unevenly spread or have holes in them). The sheets can be trimmed to   different dimensions to meet other chromatographic needs.   
- Place the sheets in tanks and develop to the top edge with ddH20   overnight. Remove the tank cover and continue development for 2 to 4 hours. A   faint, yellow, oily band is usually present at the top of the sheet. Trim off   the top edge, including the oily band, and the bottom edge of the sheets to   get 14 to 16 cm x 20 cm segments.   
- Dry the sheets completely (1-2 hours). If they are dried overnight, the   chromatographic characteristics can change because of PEI breakdown.   Caution: If the sheets are placed in the freezer slightly damp, the   PEI cellulose may separate from the vinyl backing later during   chromatography.   
- Wrap the dried sheets in foil and label them with the preparation date,   the preparer's name, the cellulose lot number, and other pertinent   information. The sheets are stable for 2-4 months at -20°C. 
SYNTHESIS OF [gamma-32P]ATP
The [gamma-32P]ATP used for labeling DNA adducts can either be purchased or synthesized in the laboratory starting with carrier-free inorganic [32P]phosphate (32Pi) and adenosine diphosphate (ADP). Preparation of [gamma-32P]ATP from 32Pi is substantially less expensive than purchasing commercial [gamma-32P]ATP.

The procedure used for preparing [gamma-32P]ATP is based on the method of Gupta et al. (1982) and Gupta and Randerath (1988) (also see Johnson and Walseth (1979) for the original method). We prepare a synthesis premix containing all of the components for making [gamma-32P]ATP, except the 32Pi. The premix is stable for 2-3 months at -80°C. Preparation of [gamma-32P]ATP can then be easily accomplished by adding the synthesis premix to carrier-free sodium [32P]phosphate.
Preparation and Storage of [gamma-32P]ATP Synthesis Premix
To ensure the highest possible [gamma-32P]ATP specific activity (curies of 32P/mmol of ATP):
- Soak all glassware, pipette tips, microcentrifuge tubes and any other   objects that come in contact with the chemicals and enzymes used to make the   [gamma-32P]ATP synthesis premix in double-distilled, deionized   water for several hours to remove nonradioactive phosphates. When   nonradioactive phosphate is present, it will also be used to phosphorylate the   ADP along with the radioactive phosphate, thus lowering the specific activity   of the synthesized [gamma-32P]ATP.   
- Keep all components on ice when preparing the ATP synthesis premix and   immediately freeze the aliquoted ATP synthesis premix at -80°C. This   precaution will yield a product with a consistently high level of specific   activity. 

The following stock solutions are needed for preparation of the ATP synthesis premix: enzyme premix, reagent solution, and buffer solution. Keep all these solutions on ice! Enzyme (footnote 2) premix (A):
200 μL of glycerol-3-phosphate dehydrogenase (2 mg/mL)2 μL of   triosephosphate isomerase (2 mg/mL)40 μL of glyceraldehyde-3-phosphate   dehydrogenase (10 mg/mL)4 μL of 3-phosphoglycerate kinase (10 mg/mL)40   μL of lactate dehydrogenase (5 mg/mL)
Reagent solution (B):
62.5 μL of 2 mM ADP (footnote   3) (add last when making solution B)62.5 μL of 4.4   mg/mL sodium pyruvate150 μL of 0.1 M dithiothreitol250 μL of 0.5 M   Tris, pH 9.0125 μL of 2.4 mM l-glycerol-3-phosphate125 μL of 10 mM   ß-NAD+100 μL of 0.3 M   MgCl2-------------------------------------875 μL Total   volume
Buffer solution (C):
42 μL of 0.1 M dithiothreitol21 μL of 0.5 M Tris, pH   9.0375 μL of   ddH20-------------------------------------438 μL Total   volume
The [gamma-32P]ATP synthesis premix is prepared from the stock solutions as follows:
- Place 30 μL of enzyme premix A (shake to resuspend the enzymes before   taking an aliquot) in a 1.5 mL microcentrifuge tube and centrifuge at 14000   rpm for 5 minutes. Carefully remove supernatant with a pipette. The ammonium   ions in the supernatant can inhibit the T4-polynucleotide kinase used to   phosphorylate the xenobiotic-DNA adducts.   
- Add 400 μL of buffer solution C to dissolve the precipitated enzymes in   the microcentrifuge tube. Next add a 220 μL aliquot of this mixture to 875 μL   of reagent solution B and vortex. It is critical that this solution is   kept on ice.   
- Add 70 μL of the premix to labeled 0.5 mL microcentrifuge tubes held on   ice. The premix aliquots are immediately stored at -80°C. 

[gamma-32P]ATP Synthesis Procedure

Caution: When ordering 32Pi from the manufacturer request a small delivery volume (100 μL or less), otherwise the synthesized [gamma-32P]ATP may be too dilute. 

The acid-free/carrier-free 32Pi sometimes arrives contaminated with 32Pi-polyphosphates formed from catenation of 32Pi-monophosphates. The presence of 32Pi-polyphosphates will reduce the quality of the autoradiograms obtained. To eliminate this problem before [gamma-32P]ATP synthesis, the 32Pi solution from the supplier is acid-treated with 0.1 volume of 0.1 N hydrochloric acid (HCl) for 2 hours at room temperature, followed by addition of 0.055 volume of 0.2 M Tris base. One can order 32Pi in dilute HCl; however, the acid concentration is variable and this can substantially affect the pH of subsequent enzyme reactions.
- To start synthesis, 50 μL of premix is added to 5 millicuries (mCi) of   carrier-free 32Pi in a 100 μL of solution. Vortex and   let stand for 1 hour at room temperature. The mixture is vortexed once or   twice during the synthesis period.Note. New England Nuclear sends   their 32Pi (NEX 053) in a lucite container surrounded   with lead that provides a substantial level of radiation protection and this   container can be used for the synthesis and storage of the   [gamma-32P]ATP. If less than 5 mCi of   32Pi is used, then the volume of premix added is   proportionally adjusted to the amount of 32Pi used in   the [gamma-32P]ATP synthesis.   
- After synthesis it is necessary to determine if the reaction has gone to   completion. A 0.1 to 0.3 μL aliquot of the reaction mixture is spotted on a   PEI-cellulose sheet and developed in a lithium chloride (LiCl) solution (1.3 M   LiCl for commercial sheets or 1 M LiCl for laboratory prepared sheets).   
- After development in LiCl, the chromatogram is dried and exposed to   autoradiographic film. A 10 second exposure at room temperature is usually   sufficient. There should be one strong spot due to [gamma-32P]ATP   with an Rf (Rf = distance of the spot from the   start/distance of the solvent front from the start) of 0.4 to 0.5 and a faint   spot may be present from unreacted 32Pi at an   Rf of 0.9 (see  Fig.   2a ). This synthesis reaction normally goes to greater than 98% completion.   If the autoradiogram indicates that a substantial amount of   32Pi is still present, then vortex the   [gamma-32P]ATP solution and let it stand for an additional 0.5 hour   and/or increase the temperature to 37°C (Talaska et al. 1992). Reanalyze for   reaction completion as described above. Usually this reaction will yield   [gamma-32P]ATP with a specific activity of 2000 to 3000 Ci per mmol   ATP. The [gamma-32P]ATP should be used within a few days (2-3) for   labeling of DNA adduct samples. 
Determination of [gamma-32P]ATP Specific Activity
The specific activity of the [gamma-32P]ATP is determined by labeling a known amount of 2'-deoxyadenosine-3'-monophosphate with [gamma-32P]ATP and separating the products by one dimensional chromatography. The 3',(32P)5'-deoxyadenosine bisphosphate spot is located by autoradiography and then quantitated by either excising the spot and liquid scintillation spectrometry or by storage phosphor imaging (see section on storage phosphor imaging).
specific activity =Ci of 32P associated with the       2'-deoxyadenosine-bisphosphate spot/mmol of       2'-deoxyadenosine-3'-monophosphate labeled.
Reagents

Dissolve a small amount of 2'-deoxyadenosine-3'-monophosphate (Sigma D-3014) in double distilled, deionized water. Determine the concentration at neutral pH by measuring the absorbence at 260 nm and using a molar extinction coefficient of 15400 liter/moles for a 1 cm path length (concentration = absorbence/extinction coefficient). Based on the absorbence values obtained dilute the solution to 1x10-4 M. Aliquot this solution into 0.5 mL microcentrifuge tubes and store at -80°C. This solution is stable for several months at -80°C.

Assay Protocol
- A 10 μL aliquot of the 1x10-4 M   2'-deoxyadenosine-3'-monophosphate solution is diluted to 10 mL with   ddH2O to give a final concentration of 1x10-7 M.   
- To four replicate 0.5 mL microcentrifuge tubes add the following: 5 μL of   1x10-7 M 2'-deoxyadenosine-3'-monophosphate5 μL of a   [gamma-32P]ATP labeling solution made by combining 3 μL of 33   μCi/μL [gamma-32P]ATP (each replicate requires at least 20 μCi of   [gamma-32P]ATP), 10 μL of kinase buffer (0.1 M bicine, 0.1 M   MgCl2, 0.1 M dithiothreitol, 10 mM spermidine, pH 9.0), 10 μL of   ddH2O and 1 μL of 30 units/μL T4-polynucleotide kinase.   Keep on ice.   
- The samples are mixed and incubated for 40 minutes at 37°C.   Because small volumes (<25 μL) are difficult to mix adequately by   vortexing or shaking, one should centrifuge the tubes to bring all liquid to   the tube bottom and then mix the samples with a micropipettor.   
- After incubation, add 390 μL of H2O to each replicate tube, mix   with pipettor and spot 10 μL from each tube on a PEI-cellulose sheet.   
- Develop the chromatogram in 0.3 M ammonium sulfate, 10 mM sodium   phosphate, pH 7.4. Use 80% strength for laboratory prepared sheets. The   Rf values are approximately 0.15 for [gamma-32P]ATP, 0.4   for 3'-,(32P)5'-deoxyadenosine bisphosphate and 0.9 for inorganic   phosphate ( Fig.   2b ).   
- After chromatography, locate the 3'-,(32P)5'-deoxyadenosine   bisphosphate spot by autoradiography.   
- Excise the spot, place it in a liquid scintillation vial and count by   liquid scintillation spectrometry. The 3'-,(32P)5'-deoxyadenosine   can also be quantitated on the chromatogram by storage phosphor imaging. 

Sample Calculation for Determining Specific ActivityThe following information is needed: amount of 2'-deoxyadenosine-3'-monophosphate labeled in moles (i.e. 5x10-13 moles), the time at which the 3',(32P)5'-deoxyadenosine bisphosphate spots were counted (i.e., 8/1/93 at 800 hours), the average disintegrations per minute (dpm) for the four replicates (i.e., 70,398 dpm), time the DNA samples were postlabeled (i.e. 8/5/93 at 1600 hours), the dilution aliquot factor (i.e., 40 if a 10 μL aliquot of a total volume of 400 μL was spotted on a PEI sheet), and the conversion factor from dpm to curies (1 curie = 2.22x1012 dpm).

Specific activity at 1600 hours on 8/5/93 =(dpm on 8/1/93) x (decay correction) x (dilution factor)/(moles of 2'-deoxyadenosine-3'-monophosphate).
Decay correction = N/No= e (-0.693T/T1/2)T= time elapsed (decay period) T1/2= half-life for isotope used (14.3 days for     32P)N= dpm at time TNo= dpm at ToSpecific activity at the time of sample labeling   (8/5/93)= (70,398) x {e[-0.693 x       4.33d/14.3]} x (40)/(5x10-13)= 4.565x1018 dpm/mol= 2,057 Ci/mmol on 8/5/93           Table of Contents