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32P-Postlabeling Protocols for Assaying Levels of Hydrophobic DNA Adducts in FishWilliam L. Reichert and Barbara FrenchNational Marine Fisheries ServiceNorthwest Fisheries Science Center Coast Zone and Estuarine Studies Division 2725 Montlake Blvd. E. Seattle WA 98112-2097 June 1994 U.S. DEPARTMENT OF COMMERCE Ronald H. Brown, Secretary
National Oceanic and Atmospheric Administration
National Marine Fisheries Service |
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.
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.
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.
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) Sheets
A 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 Homogenizers
A 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.
Spectrophotometer
Shimadzu 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 Dewars
Used 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.
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.
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 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 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).
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.
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.
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
Preparation and Pouring of PEI-Cellulose Slurry
Cutting, Washing and Storage of PEI-Cellulose Sheets
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.
To ensure the highest possible [gamma-32P]ATP specific activity (curies of 32P/mmol of ATP):
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 pyruvate
150 µL of 0.1 M dithiothreitol
250 µL of 0.5 M Tris, pH 9.0
125 µL of 2.4 mM l-glycerol-3-phosphate
125 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 dithiothreitol
21 µL of 0.5 M Tris, pH 9.0
375 µL of ddH20
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438 µL Total volume
The [gamma-32P]ATP synthesis premix is prepared from the stock solutions as follows:
[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.
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. |
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.
Sample Calculation for Determining Specific Activity
The 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 T |
No | = dpm at To |
Specific 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
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