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Measurement of UVB-induced DNA damage in marine planktonic communities
23 Measurement of UVB-induced D N A Damage in Marine Planktonic Communities W a d e H Jeffrey' and David L Mitchell 2 'Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, FL 32514, USA; 2The University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis, Science Park - Research Division, Smithville, TX 78957, USA
CONTENTS
Introduction Principle and methodology Applications Data analysis Conclusions and future directions
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INTRODUCTION Ultraviolet radiation (UVR) has been recognized for many years as a potential stress for organisms in a variety of environments (Worrest at al., 1978; 1981; Calkins, 1982) and as a factor in biogeochemical cycling (Zepp at al., 1995). Direct biological effects of UVR result from absorption of specific wavelengths of light by specific macromolecules and the dissipation of the absorbed energy via photochemical reactions (Mitchell, 1995). Cellular targets of UVR include nucleic acids, proteins, membrane lipids, the cytoskeleton, and photosystem 1i (Vincent and Roy, 1993; Mitchell, 1995). Dimerizations between adjacent pyrimidine bases are the most prevalent photoreactions resulting from the direct action of UVR on DNA (Mitchell, 1995). The two major photoproducts are the cyclobutyl pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidinone photoproduct [(6-4)photoproduct; Figure 23.1], which is converted to its valence photoisomer, the Dewar pyrimidinone, by absorption of UVB light between 310 and 320 nm (Mitchell, 1995). Formation and structural differences of the (6-4)/Dewar photoproducts and CPDs are significant and determine their different molecular and biological effects (Mitchell and Nairn, 1989). Both CPDs and (6-4) photoproducts can inhibit DNA synthesis and gene transcription, but because of structural differences, the (6-4) photoproduct is 300-fold more efficient at blocking the progression of DNA polymerase
METHODS IN MICROBIOLOGY, VOLUME 30 ISBN I1-12-521530 4
Copyright © 2001 Academic Press Ltd All rights of reproduction m anv form reserved
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than the CPD. Although induced at lower frequencies, the (6-4) photoproduct and Dewar isomer may be responsible for many of the lethal effects of UV-B radiation. Although most research examining the effects of UVR on marine microbial communities has been directed at phytoplankton and primary production, it is now apparent that all microbial trophic levels must be considered when investigating the ecological impact of UVR. The importance of bacterioplankton in oceanic processes has become widely recognized. Bacteria have been found to play a vital role in carbon cycling, transferring significant amounts of material to higher organisms. Numerous studies have shown that bacterial production is of the same order of magnitude as primary production. Although the data are quite variable and depend on location and season (Fuhrman and Azam, 1980; Hansen at al., 1983; Sullivan et al., 1990; Cota at al., 1990), bacterial production values often equal 50% of the primary production. Bacteria have been found to account for up to 90~ of the cellular DNA in oceanic environments (Paul and Carlson, 1984; Paul et al., 1985; Coffin et al., 1990) and the role of bacteria in elemental and nutrient cycling has received extensive study (Falkowski and Woodham, 1992). Bacteria, in concert with their predators (viruses and eukaryotes), play critical roles in nutrient recycling in the water column. It is plausible that reduced bacterial nutrient cycling resulting from UVB injury may result in diminished primary production. Likewise, it might be expected that a decrease in phytoplankton production may result in a decline in bacterial 470
production which may be compounded by direct UVB effects on bacterioplankton. UVR may directly impact viruses, bacteria, phytoplankton or zooplankton via direct DNA damage and reduced rates of production. Direct effects on one trophic group mav result in an indirect impact on others. The effects of UVR on marine bacterioplankton has been most often investigated by using radiolabeled precursor molecules such as ~Hthymidine (TdR; Fuhrman and Azam, 1982) and ~H- or '~C-leucine (Leu; Chin-Leo and Kirchman, 1988; Simon and Azam, 1989). Broad-band cutoff filters (e.g. Mylar 500D, UF-3 plexiglass) have then been used to selectively exclude UVB or UVB+UVA. The rate of incorporation of radiolabeled substrate is compared among treatments and compared relative to a control sample incubated in the dark. Using this method, Herndl et al. (1993) demonstrated a 48% inhibition of TdR incorporation, relative to a dark control, in surface water samples taken from the northern Adriatic Sea after 4 h incubations. Aas et al. (1996) sought to further define the spectral sensitivity of TdR and Leu incorporation in natural bacterial populations. Incubations with TdR and Leu of surface waters from a mesotrophic estuary were performed on six separate occasions. Following exposure for 6.5 to 9 h, significant differences in inhibition of incorporation of TdR and Leu relative to a dark control for treatments in full sunlight and with UVB excluded occurred. Full sunlight and UVB inhibited TdR incorporation by 44%, and 39(7,, respectively. In contrast, Leu incorporation was inhibited 29% by full sunlight but 83(,;~ of this inhibition was due to UVB. Since bacterial production is inhibited significantly by UVB, it is likely that a major cause of this inhibition is direct damage to DNA. It has been shown that bacterioplankton may experience significant amounts of DNA damage (CPDs) in surface waters, often twice the amount of larger eukaryotic cells (Jeffrey et al., 1996a). Bacterioplankton have been shown to accumulate DNA damage over a solar day and to repair the majority of that damage during the night in the Gulf of Mexico (Jeffrey et al., 1996a,b). DNA damage may extend to depths of 10 in or more in calm waters but the amount of damage may be significantly altered by surface water mixing events (Jeffrey et al., 1996a; 1997). Radioimmunoassay (RIA) is a competitive binding assay between an unlabeled and radiolabeled antigen for binding to an antibody raised against that antigen. We have adapted this technique to the measurement of specific DNA photoproducts in the DNA of UV-irradiated cells (Mitchell and Clarkson, 1981; Mitchell, 1996). The following description is given for quantification of CPDs and (6-4) photoproducts in DNA using RIA. For convenience, the radiolabeled antigen is referred to as the 'probe' and the unlabeled competitor as 'sample' or 'standard'. The arnount of radiolabeled antigen bound to the antibody is determined by separating the antigen-antibody complex from free antigen by, for example, secondary antibody or high salt precipitation. The amount of radioactivity in the antigen-antibody complex in the presence of known amounts of competitor (i.e. standards) can then be used to quantify the amount of unknown sample present in the reaction. Under these conditions, 471
antibody binding to an unlabeled competitor (sample DNA) results in reduced binding to the radiolabeled ligand (i.e. inhibition). Samples are compared to results obtained with standard DNA (i.e. pUC19 plasmid DNA) irradiated (i.e. using UVC light) such that the frequency of photoproduct formation is known. DNA damage results are reported per unit (megabase) DNA and are therefore independent of the concentration of DNA present in the original sample or the amount of DNA assayed. The sensitivity of the RIA is determined by the affinity of the antibody and specific activity of the radiolabeled antigen (probe). Using high affinity antibody and probe labeled to a high specific activity, the reaction can be limited to such an extent that extremely low levels of damage in sample DNA can be detected.
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PRINCIPLE AND METHODOLOGY Commercial sources available for the purchase of anti-UV DNA antisera include Kamiya Biomedical Co., Thousand Oaks, CA (CPD monoclonal antibody) and Dermigen, Inc., Smithville, TX [CPD, (6-4) photoproduct, and custom rabbit antisera]. The following is a procedure for producing polyclonal antisera in rabbits. The following factors determine the affinity of polyclonal antisera in rabbits: (1) the 'foreigness' of the immunogen to the host (e.g. steric and distortive deviations from normal DNA structure); (2) the number of antigenic determinants presented to the host immune system (i.e. concentration, dose); (3) accessibility of the host immune system to the antigen (e.g. single or double strandedness of the DNA); (4) stability of the immunogen in the host animal; and (5) host variability. UVC radiation (240-290nm) produces multiple types of dimeric damage in DNA, predominantly the CPD, (6-4) photoproduct, and Dewar pyrimidinone (Cadet and Vigny, 1990). In a mixture of antigenic determinants, the lesion with the greatest immunogenicity will elicit the greatest immune response. The (6-4) photoproduct, which bends the DNA helix approximately 42 ° elicits a much greater response in rabbits than the CPD which bends DNA only about 7 °. The anti-(6-4)photoproduct subpopulation displays about 10- to 100-fold more affinity than the CPD. Because of this, UVC-DNA antisera can be diluted to such an extent that binding to the minor CPD antibody subpopulation is undetectable and the RIA is specific for the (6-4) photoproduct. For CPD antisera it is necessary to produce DNA containing this photoproduct exclusively. DNA is irradiated with UVB light (290-320 nm) in the presence of a triplet sensitizer (e.g. 2 x 10 ~ M acetophenone or 10~ acetone) to produce predominately cis,syn CPDs (Lamola and Yamane, 1967). The DNA is extensively dialyzed post-irradiation to remove the sensitizer. For production of anti-UV DNA antisera we typically immunize four rabbits and select the animals that show the greatest immune response for exsanguination (Figure 23.2). 472
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Equipment and reagents • 1-120 (HPLC or Millipore-filtered) • Salmon testes (or calf thymus) DNA (Sigma) • Acetone • Methylated bovine serum albumin (Sigma A1009) • DNA nick-translation kit (Boehringer-Mannheim #976 776) • 32P-labeled deoxynucleotide triphosphates (dNTPs) (NEN, Amersham, or
ICN) • TE buffer (I 0 mM Tris, pH 8.0, I mM EDTA) • Lysis buffer B [10 mM -Iris, pH 8.0, I mM EDTA, 0.5% SDS, 100 IJg ml' DNase-free RNase A (Boehringer-Mannheim #109-169)]
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• Chloroform:isoamyl alcohol (24:1)
• Tris-saturated phenol (pH > 7.6) (Boehringer-Mannheim #100-997) • 10×TES (100 mMTris, pH 8.0, 10 mM EDTA, 1.5 M NaCI) • Gelatin (Type B: bovine skin) (Sigma #G-9382) • RIA buffer ( I x T E + 0.2% gelatin)The gelatin is heated into solution using a heated magnetic stirrer (not microwave) and heated to precisely 39J,0°C. Overheating (by as much as I-2°C) will result in prohibitive background! The cause of this is unknown. • Normal rabbit sera (NRS) (Calbiochem #566442); stored frozen in 200 IA aliquots.We have found that NRS (Calbiochem) diluted 1/40 in RIA buffer is optimal for immune pellet formation. Obviously other sources are readily available, however, we suggest that each batch be titrated in a binding assay to determine the optimal dilution. • Goat anti-rabbit IgG [Calbiochem #539844 or #539845 (bull<)]; stored frozen in 0.5 ml aliquots. • Tissue solubilizer (NCS-II from Amersham; #NNCS.502) supplemented with 10% (v:v) H20. • Scintillation cocktail (e.g. ScintiSafe from Fisher) containing I ml I' acetic acid (to neutralize the tissue solubilizer).
Preparation of i m m u n o g e n C o m m e r c i a l D N A (salmon testes or calf t h y m u s from Sigma) is diluted to lmgml' in 10ml sterile H,O (as d e t e r m i n e d by optical density at 260 nm). Diluted d o u b l e - s t r a n d e d D N A is UV-irradiated using one of the following protocols. 1. The i m m u n o g e n for anti-CPD sera is p r o d u c e d by irradiating the D N A solution (1 m g ml ') diluted in 10% acetone (final concentration; v:v) with - 7 5 k J m ~ UVB light in a glass 100 m m plate. The UVB source consists of four Westinghouse FS20 s u n l a m p s filtered through cellulose acetate (Kodacel f r o m Kodak) with a w a v e l e n g t h cutoff of 290 n m (Rosenstein, 1984). D o s i m e t r y is d e t e r m i n e d with an appropriate p h o t o m e t e r / r a d i o m e t e r (e.g. ILl400 p h o t o m e t e r coupled to a SCS 280 probe). At a distance of - 10 cm the fluence rate is - 5 J m -~s ~, hence, exposure times of N 4 h are required for a d e q u a t e CPD induction. The D N A is extensively dialyzed post-irradiation to r e m o v e a n y acetone. 2. The i m m u n o g e n for anti-(6-4)PD sera is p r o d u c e d b y irradiating D N A with 60 kJ m 2 UVC light. The UVC source consists of a b a n k of five Philips Sterilamp G8T5 bulbs emitting p r e d o m i n a n t l y 254 n m light. At a distance of ~ 20 cm the fluence rate is - 14 J m ~s ' and at this fluence rate the a v e r a g e duration of exposure is - 2 h. H e a t d e n a t u r e UV-irradiated D N A at 100°C for 10 min then place on ice. Single-stranded UV-irradiated D N A is then electrostatically coupled to m e t h y l a t e d bovine s e r u m albumin. Methylated BSA is a d d e d d r o p w i s e (approximately 50 pl each drop) until the UV-irradiated D N A solution turns cloudy.
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Immunization schedule Four New Zealand White female rabbits are initially injected subcutaneously at 10 sites (100 pl each) with 0.5 ml immunogen mixed with an equal volume of Freund's Complete Adjuvant (final concentration of 0. I mg ml ' UV-DNA). Rabbits are subsequently injected using the same protocol as above at two week intervals except that Freund's Incomplete, rather than Complete, Adjuvant is mixed with 0.5 ml immunogen. At 10-12 days following the second injection I ml of serum is drawn and binding affinity evaluated using immunoprecipitation (see below). Immunization is continued at two week intervals until sufficient binding activity is attained at which time antisera (60-80 ml) are drawn from the animal using heart puncture. Antisera are dispensed into I ml aliquots and stored at -20°C. Repeated freezing and thawing of antisera is to be strictly limited since this can severely reduce binding activity.
Determination of antiserum binding using immunoprecipitation • Probe synthesis: Both CPD and (6-4)PD frequencies are greatest in nucleic acid substrates containing a high A+T:G+C ratio. Hence, optimal substrates for the radiolabeled probe include Clostridium perfringens D N A as well as the homopolymer poly(dA):poly(dT). Nick-translate D N A (0. I lag) with ~2P-dCTP and/or 3~P-TTP to give a specific activity of - 5 × 108-109cpm lag ' (BoehringerMannheim NickTranslation Kit #976 776).A typical reaction includes 2 pl 10× buffer (from kit); 2 lal dATP [for poly(dA):poly(dT)]; or 2 tJI each dATP and dGTP (for DNA); 0.5 lal poly(dA):poly(dT) or C. perfringens D N A (diluted to 20 lug per 100 lal); 12.5 la132P-TTP at 10 mCi ml ' (NEN orAmersham); 3 4 lal DNasel/DNA polymerase I enzyme mix (from kit). Incubate for 3 0 4 5 min at 15°C. Separate radiolabeled ligand from free dNTPs using a Nick Column (Pharmacia) eluting the ligand with TE buffer. • Irradiate 32P-labeled probe with 30 kJ m 2 UVC light and restore the volume if necessary (due to evaporation) with H20 and dilute 2500- to 5000-fold in RIA buffer (yielding 2.5-5.0 pg probe in 50 pl buffer).The amount of probe added to the RIA determines its sensitivity. It is essential to use 10 pg or less and have enough radioactivity in the assay (cpm) to yield useful binding (and inhibition) data. Hence, if a 5000 dilution of probe leaves < 500 cpm in 50 lal, a greater concentration must be used. Good assay conditions should be limited to at least 500 cpm per 50 lal (added to reaction) at a probe dilution not to exceed I/I 250. • Add I ml of RIA buffer to duplicate 12 mm disposable culture tubes. Add 50 lal antiserum diluted in RIA buffer at half-log increments from 1/1000 to I/I 000 000 (dilution prior to dispensing). Duplicate tubes without antiserum are dispensed to determine background. Add 50 pl of diluted ~2P-labeled probe and vortex well. Incubate for 3 4 h with gentle rotation (optional) in a 37°C dry incubator. • Separately add 50 lal of normal rabbit serum diluted 1/40 in RIA buffer and 50 lal of goat anti-rabbit IgG diluted 1/20 and vortex well. Incubate at 4°C for 2 days until immune pellet (translucence) develops. Centrifuge tubes at - 35004000 rpm for 30 to 45 rain. Decant supernatant, invert tubes onto absorbant paper in
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test tube rack, and drain for 5 min. Wipe the lip of the tube with a cotton applicator covered with a tissue to remove any accumulated liquid. Add 100 lul of NCS Tissue Solubilizer (Amersham) supplemented with 10% H20 and incubate at 37°C (or room temperature) with rotation until immune pellet is completely dissolved. It is extremely important that the immune pellet be completely solubilized but not allowed to dry. Partial solubilization will result in bad duplicates. Add 2 ml of scintillation cocktail (e.g. ScintiSafe from Fisher) supplemented with I ml I ' acetic acid (to eliminated chemoluminescence generated by tissue solubilizer) and vortex.The samples can either be decanted into 20 ml scintillation vials and washed twice with 4 ml additional scintillation cocktail or the RIA tubes can be placed directly into scintillation vials and counted. Count 32p using liquid scintillation counter.
Isolation of D N A One of the major attributes of RIA is its ability to measure photoproducts in DNA that has not been extensively purified and most DNA isolation protocols are suitable for RIA sample preparation. The protocol described below has been found to work well in a variety of marine environments. The radioimmunoassay is less dependent on DNA concentration than on the number of photolesions present. If DNA damage is low, more cells will be needed. Conversely, smaller samples may be used when damage is high. In general, we have found that a final extract of 2-4 pg DNA is sufficient to detect damage in marine planktonic communities, although 5-10 pg is preferable. This allows replicate analysis for each photoproduct as well as multiple photoproducts. In open ocean samples, this may require 50-701 of seawater. In coastal waters, 51 is often sufficient. In general, samples are collected by filtration. Size fraction filtration may be used to isolate particular members of the microbial community (e.g. bacterioplankton <0.8 l~m, >0.21.lm pore size). We have found Gelman SUPOR (polysulfone) filters with 142 mm diameters to work well. Filters are collected, folded, and placed in 2 oz polyethylene bags. Filters are stored at -80°C until extraction. To extract DNA remove the filter from the freezer and immediately crush the filter in the bag before it thaws. Filter pieces were then poured into a 50 ml Oak Ridge centrifuge tube. • Add 5 ml of STE (I 0 mM Tris, pH 8; I mM EDTA; I00 mM NaCI) containing 1% SDS (sodium dodecyl sulfate) to each tube. Cap, vortex for 15 s, and place in a boiling water bath for 5 rain. • Aspirate the lysate and place in a new centrifuge tube.Wash the filter pieces with an additional 5 ml of STE, vortex for 15 s, and combine with the lysate. • Extract with I 0 ml of chloroform:isoamyl alchohol (24:1). Collect the aqueous phase after centrifuging at 4°C for 30 min at 3000 rpm. • Decant the aqueous phase into a new Oak Ridge tube, add I 0 lal of a 25 mg ml ' glycogen solution, 0. I volume of 3 M sodium acetate (pH 5), and an equal volume of isopropanoI.Vortex and precipitate the sample overnight at -20°C. • Collect the precipitate by centrifugation at 12000g and 4°C for 30 rain. Decant the supernatant and wash the pellet with 10 ml cold 70% ethanol. Centrifuge at 12 O00g at 4°C for I 0 min. Decant the supernatant.
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• Re-suspend the pellet in I ml of STE before transferring to a 2 ml microfuge tube. Re-precipitate as above, dry the pellet then re-suspend in 200 pl STE. DNA concentrations are determined spectrofluorometrically (Paul and Myers, 1982) using either a Picogreen (for native DNA) or Oligreen (for denatured DNA) reagent from Molecular Probes (Eugene, OR).We prefer the Oligreen method since it quantifies DNA concentrations immediately prior to analysis.
Competitive binding assay (RIA) The RIA is s i m p l y the basic i m m u n o p r e c i p i t a t i o n reaction outlined a b o v e into which a standard or s a m p l e D N A has been a d d e d to c o m p e t e with the radiolabeled p r o b e for a n t i b o d y binding. Hence, the p r o c e d u r e is exactly the s a m e as that used for i m m u n o p r e c i p i t a t i o n with the following additions/modifications. • A single dilution of antiserum is used. This dilution is determined from immunoprecipitation analyses of binding activity (see above) and should yield 30-60% of the radiolabeled probe in the immune pellet. • For quantification of CPDs or (6-4)PDs a dose response of heat-denatured salmon testes (Sigma) UV-irradiated DNA is used as standard (Table 23. I).We routinely use doses of 3, I 0, 30, 100, and 300 J m 2 as our standard curve and assay the same amount of standard as sample DNA.We have determined rates of photoproduct induction using independent analysis of CPDs as T4 UV endonuclease-sensitive sites and (6-4) photoproducts as photoinduced alkalilabile sites (Mitchell et al., 1990). From these values the UVC doses are converted to photoproduct frequencies using 8. I CPDs and 1.56 (6-4) photoproducts per megabase DNA per joule m 2.When relative, rather than exact, amounts of CPDs or (6-4)PDs are adequate for experimental purposes (as in DNA repair experiments) the sample harvested at the time of irradiation can be diluted in half-log increments to generate a standard. From this type of standard curve data representing percentage photoproducts remaining (or repaired) can be generated. • Unlabeled competitor mammalian DNA, radioactive ligand, and diluted antibody are incubated together for 3 h at 37°C with gentle rotation (optional). (As above, it is prudent to perform a preliminary titration of sample DNA to determine the amount required for adequate inhibition in the RIA).The total volume of sample DNA added can vary within certain limitations. Sample volumes <100 [JI do not significantly effect the reaction conditions (e.g. total binding). Sample volumes > 100 pl can be used, however, the total reaction volume should be increased accordingly (i.e. doubled).
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APPLICATIONS We h a v e used the described m e t h o d to s t u d y D N A d a m a g e in a wide variety of systems, from the Southern Ocean (Jeffrey et al., 1997) to coral reef microbial c o m m u n i t i e s (Lyons et al., 1998). Tlle protocol w o r k s
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equally well for bacterioplankton, phytoplankton (Karentz et al., 1991), and fish larvae (Malloy et al., 1997). Most often, the technique has been used to determine the amount of UVB-induced DNA damage in microbial communities (Jeffrey et al., 1996a,b). This has been done as a function of time of day, depth in the water column, cell size fraction, or seasonal comparisons. It is also possible to examine rates of DNA damage repair by incorporating broadband spectral filters (e.g. Mylar 500 D or acrylics, Aas et al., 1996) post-UVB irradiation. The main obstacle to doing this with marine microbial communities is the large volume required to be filtered for each time point. We have had some success by constructing UV-transparent incubators that will hold up to 200 1of seawater at ambient temperatures from which samples may be collected over time. High cell densities in laboratory cultures allow more detailed experimentation to be designed (Joux et al., 1999). The DNA damage assays are identical, with only minor modifications in the DNA extraction protocol required. The kinetics of DNA damage induction and repair may be determined as a function of dose response, for instance, and comparisons made between different strains. Smaller volumes may allow greater spectral resolution of a UV response by incorporating additional optical filters (e.g. Schott filters, Schott Glass Technologies, Duryea, PA) which are not available in sizes needed to incubate seawater samples.
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DATA ANALYSIS A sample Excel spreadsheet for quantification of CPDs or (6-4)PDs is shown in Table 23.1. A sample Excel spreadsheet for quantification of relative photoproducts remaining at specific times post UV-irradiation (e.g. in a DNA repair experiment) is shown in Table 23.2.
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CONCLUSIONS
AND FUTURE DIRECTIONS
Different types of DNA damage have different immunogenicities and may not be suitable for the production of antibodies in rabbits or mice. We have been unsuccessful in raising rabbit antisera against DNA irradiated with UVA light, DNA cross-linked with mitomycin C, or DNA homopolymers or alternating copolymers irradiated with UVC light. These failures may be due to the low antigenicity of the damage itself (e.g. UVA, mitomycin C), immunogen stability in the host (e.g. digestion of oligonucleotides or polynucleotides), or host variability (i.e. not enough rabbits). We have been successful, however, at raising polyclonal antibodies against UVC light, triplet-sensitized UVB light, DNA containing acetylaminofluorene adducts, DNA containing benzo[a]pyrene diolepoxide adducts, DNA treated with osmium tetroxide (i.e. thymine glycols), and HPLC-purified 8-oxodeoxyguanosine covalently linked to a protein hapten. 480
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RIA and ELISA are p o w e r f u l techniques for the sensitive and specific detection of genotoxic d a m a g e in DNA. Other techniques, such as quantitative i m m u n o c y t o c h e m i s t r y , immunohistochemistry, and i m m u n o e l e c tron m i c r o s c o p y have been designed to detect D N A d a m a g e in situ, thus visualizing the distribution of d a m a g e in tissues and cells. D a m a g e d D N A m a y be separated from u n d a m a g e d D N A fragments b y i m m u n o precipitation, in-tmunoaffinity chromatography, or nitrocellulose binding. Enrichment of d a m a g e d D N A f r a g m e n t s using antibodies has been c o m b i n e d with PCR amplification and Southern analysis to determine the genomic distribution of D N A d a m a g e (Hochleitner et al., 1991) and with HPLC (or ligation-mediated PCR) to increase the resolution and, hence, sensitivity of detection ( G r o o p m a n et al., 1992).
References Aas, P., Lyons, M., Pledger, R., Mitchell, D. L. and Jeffrey, W. H. (1996). Inhibition of bacterial activities by solar radiation in nearshore waters and the Gulf of Mexico. Aquatic Microbial Ecol. 11, 229-238. Cadet, J. and Vigny, P. (1990). The photochemistry of nucleic acids. In: Biooor~aJTic PhotochenlistJ 7, Vol. 1: Ptlotochenlistry and the Nuch'ic Acids (H. Morrison, Ed.), pp. 1-272. John Wiley and Sons, New Yurk. Calkins, J. (Ed.) (1982). The Roh' qf Solar Llltravi~det RadiatioH iH Marine Ecosystems. Plenum Press, New York. Chin-Leo, G. and Kirchman, D. L. (1988). Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine. Appl. Euviron. Micmbiol. 54, 1934 1939. Coffin, R. B., Velinsky, D., Devereux, R., Price, W. A. and Cifuentes, L. (1990). Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria. Appl. Euvirol~. Micmbiol. 56, 2012-2020. Cota, G. E, Kottmeier, S. T., Robinson, D. H., Smith, W. O. and Sullivan, C. W. (1990). Bacterioplankton in the marginal ice zone of the Weddel] Sea: biomass, production and metabolic activities during Austral summer. Deep-Sea Res. 37, 1145-1167. Falkowski, P. G. and Woodham, A. D. (1992). Prima12t/Production aud Biogeochemical Cycles in the Sea. Plenum Press, New York. Fuhrman, J. A. and Azam, E (1980). Bacterioplankton secondary production estimates for coastal waters of British Columbia, Antarctica, and California. App[. El~virou. Microbiol. 39, 1085 1095. Fuhrman, J. A. and Azam, E (1982). Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: evaluation and field results. Mar. Biol. 66, 109-120. Groopman, J. D., Zhu, J. Q., Donahue, P. R., Pikul, A., Zhang, L. S., Chen, J. S. and Wogan, G.N. (1992). Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region, People's Republic of China. Ca~Tcer Res. 52, 45-52. Hansen, R. B., Sharer, D., Ryan, T., Pope, D. and Lowery, H. K. (1983). Bacterioplankton in the Antarctic ocean waters during late Austral winter, abundance, frequency of dividing cells, and estimates of production. Appl. EllviroJl. Microbiol. 45, 1622 1632. Herndl, G. ]., M/_iller-Niklas, G. and Frick, J. (1993). Major role of ultraviolet-B in controlling bacterioplankton growth in the surface layer of the ocean. Nature 361, 717-719.
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Hochleitner, K., Thomale, J., Nikitin, A. Yu and Rajewsky, M. E (1991). Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences. Nucleic Acids Rcs. 19, 4467-4472. Jeffrey, W. H., Aas, P, Lyons, M. M., Pledger, R., Mitchell, D. L. and Coffin, R. B. (1996a). Ambient solar radiation induced photodamage in marine bacterioplankton. Photochenl. Photobiol. 64, 419-427. Jeffrey, W. 1t., Miller, R. V. and Mitchell, D. L. (1997). Detection of ultraviolet radiation induced DNA damage in microbial communities of the Gerlache Strait. Aittarctic J. LIS 32, 85-87. Jeffre}; W. H., Pledger, R. J., Aas, P., Hager, S., Coffin, R. B., Von Haven, R. and Mitchell, D. L. (1996b). Diel and depth profiles of DNA photodamage in bacteriuplankton exposed to ambient solar radiation. Mar'. Ecol. Prog. Ser. 137, 283-291. Joux, E, Jeffrey, W. H., Lebaron, I~ and Mitchell, D. (1999). Marine bacteria display diverse responses to u/traviolet-B radiation. Appl. Environ7. Microbiol. 65, 3820-3827. Karentz, D., Cleaver, J. E. and Mitchell, D. L. (1991). Cell survival characteristics and molecular responses of antarctic phytoplankton to ultraviolet-B radiation. J. Phycol. 27, 326-341. Lamola, A. A. and Yamane, T. (1967). Sensitized photodimerization of thymine in DNA. Proc. Natl. AcmL Sci. LISA 58, 443-446. Lyons, M. M., Aas, P., Pakulski, J. D., Van Waasbergen, L., Mitchell, D. L., Miller, R. V. and Jeffrey, W. H. (1998). Ultraviolet radiation induced DNA damage in coral reef microbial communities. Mar. Biol. 130, 537-543. Malloy, K. D., Holman, M. A., Mitchell, D. and Detrich, H. W. III (1997). Solar UVBinduced DNA damage and photoenzymatic repair in Antarctic zooplankton. Proc. N~ttl. Acad. Sci. USA 94, 1258-1263. Mitchell, D. L. (1995). Ultraviolet radiation damage to DNA. In: Molecular Biology amt Bioteclmology: A Comptvhensive Desk Reference (R. A. Meyers, Ed.), pp. 939-943. VCH Publishers, New York. Mitchell, D. L. (1996). Radioimmunoassay of DNA damaged by ultraviolet light. In: Techllologics (or Detectiol~ qf DNA Damage ~TmtMutatiotzs (G. Pfeifer, Ed.), pp. 73-85. Plenum, New York, Mitchell, D. L. and Clarkson, J. M. (1981). The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA. Biochim. Biophys. Acta 655, 54 60. Mitchell, D. L. and Nairn, R. S. (1989). The biology of the (6-4) photoproduct. Photochem. Photobiol. 49, 805-819. Mitchell, D. L., Brash, D. E. and Nairn, R. S. (1990). Rapid repair of pyrimidine (6-4)pyrimidone photoproducts in human cells does not result from change in epitope conformation. NHcleic Acids Res. 18, 963-971. Paul, J. H. and Carlson, D. (1984). Genetic material in the marine environment: implication for bacterial DNA. Limm~l. Oceam~gr. 29, 1091-1097. Paul, J. H. and Myers, B. (1982). Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258. Appl. E1~virott. Microbiol. 43, 1393-1399. Paul, J. H., Jeffrey, W. H. and Deflaun, M. E (1985). Particulate DNA in subtropical oceanic and estuarine planktonic environments. Mar. Biol. 90, 95-101; Appl. EHvirotz, Microbio[. 43, 1393-1399. Rosenstein, B. S. (1984). Photoreactivation of ICR 2A frog cells exposed to solar UV wavelengths. Photochcm. Photobiol. 40, 207-213. Simon, M. and Azam, E (1989). Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Pro~,,. Set. 51,201-213.
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Sullivan, C. W., Cota, G. E, Krempin, D. W. and Smith, W. O. Jr. (1990). Distribution and activity of bacterioplankton in the marginal ice zone of the Weddell-Scotia Sea during austral spring. Mar. Ecol. Prog. Set. 63, 239-252. Vincent, W. E and Roy, S. (1993). Solar ultraviolet-B radiation and aquatic primary production: damage, protection, and recovery. Environ. Rev. 1, 1-12. Worrest, R., Dyke, H. V. and Thomson, B. (1978). Impact of enhanced simulated solar ultraviolet radiation upon a marine community. Photochem. Photobiol. 27, 471-478. Worrest, R., Wolniakowski, K., Scott, J., Brooker, D. and Dyke, H. V. (1981). Sensitivity of marine phytoplankton to UV-B radiation: impact upon a model ecosystem. Photochem. Photobiol. 33, 223-227. Zepp, R., Callaghan, T. and Erickson, D. (1995). Effects of increased solar ultraviolet radiation on biogeochemical cycles. Ambio 24, 181-187.
List of suppliers Amersham Pharmacia Biotech, Inc. 800 Centennial Avemte P.O. Box 1327 Piscataway, NJ 08855-1327, USA Tel: 732-457-8000 Fax: 732-457-0557 Radio-labeled dNTPs; Tissue solubilizer
Eastman Kodak Company Kodacel Marketing Roll Coating Division 1669 Lake Avenue Kodak Park, Building 21, Floor 2 Rochestel; NY 14652-3202, USA Teh 800-367-5235 Fax: 716-477-3465 Kodacel
Boehringer-Mannheim E Hoffmann-La Roche Ltd CH-4070 Basel Switzerland Teh ++41 / 61 688 11 11 Fax: ++41 / 61 691 93 91
Fisher Scientific 585 Alpha Drive Pittsburgh, PA 15238, USA Tel: 800-766-7000
Nick Translation kit; DNase-free RNAse A; Tris saturated phenol
Gelman Sciences (Pall Gelman Sciences) 600 S. Wagner Road Ann Arbot, M148103, USA Tel: 800-521-1360 Fax: 800-913-6440
Liquid scintillation cocktail
Calbiochem-Novabiochem Corporation 10394 Pacific Center Court San Diego, California 92121, USA Mailing Address: P.O. Box 12087 La Jolla, California 92039-2087, USA Teh 858- 450-9600 Fax: 858- 453-3552 N o r m a l rabbit sera; Goat antirabbit (IgG)
484
Supor filters
International Light, Inc. 17 Graf Road Newbu ryport, MA 01950, USA Tel: 978-465-5923 Fax: 978-462-0759 UV r a d i o m e t e r
Pharmacia and Upjohn 100 Route 206 North Peapack, New Jersey 07977, USA Tel: 908-901-8000; 888-768-5501 Fax: 908-901-8379
Millipore Corp. 186 Middlesex Turnpike Burlington, MA 01803, USA Tel: 781-533-6000
HPLC water
Nick Column Molecular Probes PO Box 22010 Eugene, OR 97402-0469, USA 4849 Pitchford Avenue Eugene, OR 97402-9165, USA Teh 541- 465-8300 Fax: 541-344-6504
Sigma Chemical 3050 Spruce Street St. Louis, MO 63103, USA Tel: 314-771-5765
DNA; methylated BSA; gelatin
Oligogreen nucleic acid dye New England Nuclear (NEN) NEN® Life Science Products, Inc. 549 Albany Street Boston, MA 02118-2512, USA Teh 800-551-2121 or 617-482-9595 Fax: 617-482-1380
Radiolabeled dNTPs
485
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CONTENTS
Introduction Principle and methodology Applications Data analysis Conclusions and future directions
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INTRODUCTION Ultraviolet radiation (UVR) has been recognized for many years as a potential stress for organisms in a variety of environments (Worrest at al., 1978; 1981; Calkins, 1982) and as a factor in biogeochemical cycling (Zepp at al., 1995). Direct biological effects of UVR result from absorption of specific wavelengths of light by specific macromolecules and the dissipation of the absorbed energy via photochemical reactions (Mitchell, 1995). Cellular targets of UVR include nucleic acids, proteins, membrane lipids, the cytoskeleton, and photosystem 1i (Vincent and Roy, 1993; Mitchell, 1995). Dimerizations between adjacent pyrimidine bases are the most prevalent photoreactions resulting from the direct action of UVR on DNA (Mitchell, 1995). The two major photoproducts are the cyclobutyl pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidinone photoproduct [(6-4)photoproduct; Figure 23.1], which is converted to its valence photoisomer, the Dewar pyrimidinone, by absorption of UVB light between 310 and 320 nm (Mitchell, 1995). Formation and structural differences of the (6-4)/Dewar photoproducts and CPDs are significant and determine their different molecular and biological effects (Mitchell and Nairn, 1989). Both CPDs and (6-4) photoproducts can inhibit DNA synthesis and gene transcription, but because of structural differences, the (6-4) photoproduct is 300-fold more efficient at blocking the progression of DNA polymerase
METHODS IN MICROBIOLOGY, VOLUME 30 ISBN I1-12-521530 4
Copyright © 2001 Academic Press Ltd All rights of reproduction m anv form reserved
DNA photoproduct formation
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than the CPD. Although induced at lower frequencies, the (6-4) photoproduct and Dewar isomer may be responsible for many of the lethal effects of UV-B radiation. Although most research examining the effects of UVR on marine microbial communities has been directed at phytoplankton and primary production, it is now apparent that all microbial trophic levels must be considered when investigating the ecological impact of UVR. The importance of bacterioplankton in oceanic processes has become widely recognized. Bacteria have been found to play a vital role in carbon cycling, transferring significant amounts of material to higher organisms. Numerous studies have shown that bacterial production is of the same order of magnitude as primary production. Although the data are quite variable and depend on location and season (Fuhrman and Azam, 1980; Hansen at al., 1983; Sullivan et al., 1990; Cota at al., 1990), bacterial production values often equal 50% of the primary production. Bacteria have been found to account for up to 90~ of the cellular DNA in oceanic environments (Paul and Carlson, 1984; Paul et al., 1985; Coffin et al., 1990) and the role of bacteria in elemental and nutrient cycling has received extensive study (Falkowski and Woodham, 1992). Bacteria, in concert with their predators (viruses and eukaryotes), play critical roles in nutrient recycling in the water column. It is plausible that reduced bacterial nutrient cycling resulting from UVB injury may result in diminished primary production. Likewise, it might be expected that a decrease in phytoplankton production may result in a decline in bacterial 470
production which may be compounded by direct UVB effects on bacterioplankton. UVR may directly impact viruses, bacteria, phytoplankton or zooplankton via direct DNA damage and reduced rates of production. Direct effects on one trophic group mav result in an indirect impact on others. The effects of UVR on marine bacterioplankton has been most often investigated by using radiolabeled precursor molecules such as ~Hthymidine (TdR; Fuhrman and Azam, 1982) and ~H- or '~C-leucine (Leu; Chin-Leo and Kirchman, 1988; Simon and Azam, 1989). Broad-band cutoff filters (e.g. Mylar 500D, UF-3 plexiglass) have then been used to selectively exclude UVB or UVB+UVA. The rate of incorporation of radiolabeled substrate is compared among treatments and compared relative to a control sample incubated in the dark. Using this method, Herndl et al. (1993) demonstrated a 48% inhibition of TdR incorporation, relative to a dark control, in surface water samples taken from the northern Adriatic Sea after 4 h incubations. Aas et al. (1996) sought to further define the spectral sensitivity of TdR and Leu incorporation in natural bacterial populations. Incubations with TdR and Leu of surface waters from a mesotrophic estuary were performed on six separate occasions. Following exposure for 6.5 to 9 h, significant differences in inhibition of incorporation of TdR and Leu relative to a dark control for treatments in full sunlight and with UVB excluded occurred. Full sunlight and UVB inhibited TdR incorporation by 44%, and 39(7,, respectively. In contrast, Leu incorporation was inhibited 29% by full sunlight but 83(,;~ of this inhibition was due to UVB. Since bacterial production is inhibited significantly by UVB, it is likely that a major cause of this inhibition is direct damage to DNA. It has been shown that bacterioplankton may experience significant amounts of DNA damage (CPDs) in surface waters, often twice the amount of larger eukaryotic cells (Jeffrey et al., 1996a). Bacterioplankton have been shown to accumulate DNA damage over a solar day and to repair the majority of that damage during the night in the Gulf of Mexico (Jeffrey et al., 1996a,b). DNA damage may extend to depths of 10 in or more in calm waters but the amount of damage may be significantly altered by surface water mixing events (Jeffrey et al., 1996a; 1997). Radioimmunoassay (RIA) is a competitive binding assay between an unlabeled and radiolabeled antigen for binding to an antibody raised against that antigen. We have adapted this technique to the measurement of specific DNA photoproducts in the DNA of UV-irradiated cells (Mitchell and Clarkson, 1981; Mitchell, 1996). The following description is given for quantification of CPDs and (6-4) photoproducts in DNA using RIA. For convenience, the radiolabeled antigen is referred to as the 'probe' and the unlabeled competitor as 'sample' or 'standard'. The arnount of radiolabeled antigen bound to the antibody is determined by separating the antigen-antibody complex from free antigen by, for example, secondary antibody or high salt precipitation. The amount of radioactivity in the antigen-antibody complex in the presence of known amounts of competitor (i.e. standards) can then be used to quantify the amount of unknown sample present in the reaction. Under these conditions, 471
antibody binding to an unlabeled competitor (sample DNA) results in reduced binding to the radiolabeled ligand (i.e. inhibition). Samples are compared to results obtained with standard DNA (i.e. pUC19 plasmid DNA) irradiated (i.e. using UVC light) such that the frequency of photoproduct formation is known. DNA damage results are reported per unit (megabase) DNA and are therefore independent of the concentration of DNA present in the original sample or the amount of DNA assayed. The sensitivity of the RIA is determined by the affinity of the antibody and specific activity of the radiolabeled antigen (probe). Using high affinity antibody and probe labeled to a high specific activity, the reaction can be limited to such an extent that extremely low levels of damage in sample DNA can be detected.
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PRINCIPLE AND METHODOLOGY Commercial sources available for the purchase of anti-UV DNA antisera include Kamiya Biomedical Co., Thousand Oaks, CA (CPD monoclonal antibody) and Dermigen, Inc., Smithville, TX [CPD, (6-4) photoproduct, and custom rabbit antisera]. The following is a procedure for producing polyclonal antisera in rabbits. The following factors determine the affinity of polyclonal antisera in rabbits: (1) the 'foreigness' of the immunogen to the host (e.g. steric and distortive deviations from normal DNA structure); (2) the number of antigenic determinants presented to the host immune system (i.e. concentration, dose); (3) accessibility of the host immune system to the antigen (e.g. single or double strandedness of the DNA); (4) stability of the immunogen in the host animal; and (5) host variability. UVC radiation (240-290nm) produces multiple types of dimeric damage in DNA, predominantly the CPD, (6-4) photoproduct, and Dewar pyrimidinone (Cadet and Vigny, 1990). In a mixture of antigenic determinants, the lesion with the greatest immunogenicity will elicit the greatest immune response. The (6-4) photoproduct, which bends the DNA helix approximately 42 ° elicits a much greater response in rabbits than the CPD which bends DNA only about 7 °. The anti-(6-4)photoproduct subpopulation displays about 10- to 100-fold more affinity than the CPD. Because of this, UVC-DNA antisera can be diluted to such an extent that binding to the minor CPD antibody subpopulation is undetectable and the RIA is specific for the (6-4) photoproduct. For CPD antisera it is necessary to produce DNA containing this photoproduct exclusively. DNA is irradiated with UVB light (290-320 nm) in the presence of a triplet sensitizer (e.g. 2 x 10 ~ M acetophenone or 10~ acetone) to produce predominately cis,syn CPDs (Lamola and Yamane, 1967). The DNA is extensively dialyzed post-irradiation to remove the sensitizer. For production of anti-UV DNA antisera we typically immunize four rabbits and select the animals that show the greatest immune response for exsanguination (Figure 23.2). 472
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Figure 23.2. Outline of the radioimmunoassay for detection of DNA damage. Antibodies are raised against a specific type of DNA lesion (e.g. CPD). Middle: binding of the antibody is characterized. Bottom: RIA is used to measure lesions in sample DNA.
Equipment and reagents • 1-120 (HPLC or Millipore-filtered) • Salmon testes (or calf thymus) DNA (Sigma) • Acetone • Methylated bovine serum albumin (Sigma A1009) • DNA nick-translation kit (Boehringer-Mannheim #976 776) • 32P-labeled deoxynucleotide triphosphates (dNTPs) (NEN, Amersham, or
ICN) • TE buffer (I 0 mM Tris, pH 8.0, I mM EDTA) • Lysis buffer B [10 mM -Iris, pH 8.0, I mM EDTA, 0.5% SDS, 100 IJg ml' DNase-free RNase A (Boehringer-Mannheim #109-169)]
473
• Chloroform:isoamyl alcohol (24:1)
• Tris-saturated phenol (pH > 7.6) (Boehringer-Mannheim #100-997) • 10×TES (100 mMTris, pH 8.0, 10 mM EDTA, 1.5 M NaCI) • Gelatin (Type B: bovine skin) (Sigma #G-9382) • RIA buffer ( I x T E + 0.2% gelatin)The gelatin is heated into solution using a heated magnetic stirrer (not microwave) and heated to precisely 39J,0°C. Overheating (by as much as I-2°C) will result in prohibitive background! The cause of this is unknown. • Normal rabbit sera (NRS) (Calbiochem #566442); stored frozen in 200 IA aliquots.We have found that NRS (Calbiochem) diluted 1/40 in RIA buffer is optimal for immune pellet formation. Obviously other sources are readily available, however, we suggest that each batch be titrated in a binding assay to determine the optimal dilution. • Goat anti-rabbit IgG [Calbiochem #539844 or #539845 (bull<)]; stored frozen in 0.5 ml aliquots. • Tissue solubilizer (NCS-II from Amersham; #NNCS.502) supplemented with 10% (v:v) H20. • Scintillation cocktail (e.g. ScintiSafe from Fisher) containing I ml I' acetic acid (to neutralize the tissue solubilizer).
Preparation of i m m u n o g e n C o m m e r c i a l D N A (salmon testes or calf t h y m u s from Sigma) is diluted to lmgml' in 10ml sterile H,O (as d e t e r m i n e d by optical density at 260 nm). Diluted d o u b l e - s t r a n d e d D N A is UV-irradiated using one of the following protocols. 1. The i m m u n o g e n for anti-CPD sera is p r o d u c e d by irradiating the D N A solution (1 m g ml ') diluted in 10% acetone (final concentration; v:v) with - 7 5 k J m ~ UVB light in a glass 100 m m plate. The UVB source consists of four Westinghouse FS20 s u n l a m p s filtered through cellulose acetate (Kodacel f r o m Kodak) with a w a v e l e n g t h cutoff of 290 n m (Rosenstein, 1984). D o s i m e t r y is d e t e r m i n e d with an appropriate p h o t o m e t e r / r a d i o m e t e r (e.g. ILl400 p h o t o m e t e r coupled to a SCS 280 probe). At a distance of - 10 cm the fluence rate is - 5 J m -~s ~, hence, exposure times of N 4 h are required for a d e q u a t e CPD induction. The D N A is extensively dialyzed post-irradiation to r e m o v e a n y acetone. 2. The i m m u n o g e n for anti-(6-4)PD sera is p r o d u c e d b y irradiating D N A with 60 kJ m 2 UVC light. The UVC source consists of a b a n k of five Philips Sterilamp G8T5 bulbs emitting p r e d o m i n a n t l y 254 n m light. At a distance of ~ 20 cm the fluence rate is - 14 J m ~s ' and at this fluence rate the a v e r a g e duration of exposure is - 2 h. H e a t d e n a t u r e UV-irradiated D N A at 100°C for 10 min then place on ice. Single-stranded UV-irradiated D N A is then electrostatically coupled to m e t h y l a t e d bovine s e r u m albumin. Methylated BSA is a d d e d d r o p w i s e (approximately 50 pl each drop) until the UV-irradiated D N A solution turns cloudy.
474
Immunization schedule Four New Zealand White female rabbits are initially injected subcutaneously at 10 sites (100 pl each) with 0.5 ml immunogen mixed with an equal volume of Freund's Complete Adjuvant (final concentration of 0. I mg ml ' UV-DNA). Rabbits are subsequently injected using the same protocol as above at two week intervals except that Freund's Incomplete, rather than Complete, Adjuvant is mixed with 0.5 ml immunogen. At 10-12 days following the second injection I ml of serum is drawn and binding affinity evaluated using immunoprecipitation (see below). Immunization is continued at two week intervals until sufficient binding activity is attained at which time antisera (60-80 ml) are drawn from the animal using heart puncture. Antisera are dispensed into I ml aliquots and stored at -20°C. Repeated freezing and thawing of antisera is to be strictly limited since this can severely reduce binding activity.
Determination of antiserum binding using immunoprecipitation • Probe synthesis: Both CPD and (6-4)PD frequencies are greatest in nucleic acid substrates containing a high A+T:G+C ratio. Hence, optimal substrates for the radiolabeled probe include Clostridium perfringens D N A as well as the homopolymer poly(dA):poly(dT). Nick-translate D N A (0. I lag) with ~2P-dCTP and/or 3~P-TTP to give a specific activity of - 5 × 108-109cpm lag ' (BoehringerMannheim NickTranslation Kit #976 776).A typical reaction includes 2 pl 10× buffer (from kit); 2 lal dATP [for poly(dA):poly(dT)]; or 2 tJI each dATP and dGTP (for DNA); 0.5 lal poly(dA):poly(dT) or C. perfringens D N A (diluted to 20 lug per 100 lal); 12.5 la132P-TTP at 10 mCi ml ' (NEN orAmersham); 3 4 lal DNasel/DNA polymerase I enzyme mix (from kit). Incubate for 3 0 4 5 min at 15°C. Separate radiolabeled ligand from free dNTPs using a Nick Column (Pharmacia) eluting the ligand with TE buffer. • Irradiate 32P-labeled probe with 30 kJ m 2 UVC light and restore the volume if necessary (due to evaporation) with H20 and dilute 2500- to 5000-fold in RIA buffer (yielding 2.5-5.0 pg probe in 50 pl buffer).The amount of probe added to the RIA determines its sensitivity. It is essential to use 10 pg or less and have enough radioactivity in the assay (cpm) to yield useful binding (and inhibition) data. Hence, if a 5000 dilution of probe leaves < 500 cpm in 50 lal, a greater concentration must be used. Good assay conditions should be limited to at least 500 cpm per 50 lal (added to reaction) at a probe dilution not to exceed I/I 250. • Add I ml of RIA buffer to duplicate 12 mm disposable culture tubes. Add 50 lal antiserum diluted in RIA buffer at half-log increments from 1/1000 to I/I 000 000 (dilution prior to dispensing). Duplicate tubes without antiserum are dispensed to determine background. Add 50 pl of diluted ~2P-labeled probe and vortex well. Incubate for 3 4 h with gentle rotation (optional) in a 37°C dry incubator. • Separately add 50 lal of normal rabbit serum diluted 1/40 in RIA buffer and 50 lal of goat anti-rabbit IgG diluted 1/20 and vortex well. Incubate at 4°C for 2 days until immune pellet (translucence) develops. Centrifuge tubes at - 35004000 rpm for 30 to 45 rain. Decant supernatant, invert tubes onto absorbant paper in
475
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test tube rack, and drain for 5 min. Wipe the lip of the tube with a cotton applicator covered with a tissue to remove any accumulated liquid. Add 100 lul of NCS Tissue Solubilizer (Amersham) supplemented with 10% H20 and incubate at 37°C (or room temperature) with rotation until immune pellet is completely dissolved. It is extremely important that the immune pellet be completely solubilized but not allowed to dry. Partial solubilization will result in bad duplicates. Add 2 ml of scintillation cocktail (e.g. ScintiSafe from Fisher) supplemented with I ml I ' acetic acid (to eliminated chemoluminescence generated by tissue solubilizer) and vortex.The samples can either be decanted into 20 ml scintillation vials and washed twice with 4 ml additional scintillation cocktail or the RIA tubes can be placed directly into scintillation vials and counted. Count 32p using liquid scintillation counter.
Isolation of D N A One of the major attributes of RIA is its ability to measure photoproducts in DNA that has not been extensively purified and most DNA isolation protocols are suitable for RIA sample preparation. The protocol described below has been found to work well in a variety of marine environments. The radioimmunoassay is less dependent on DNA concentration than on the number of photolesions present. If DNA damage is low, more cells will be needed. Conversely, smaller samples may be used when damage is high. In general, we have found that a final extract of 2-4 pg DNA is sufficient to detect damage in marine planktonic communities, although 5-10 pg is preferable. This allows replicate analysis for each photoproduct as well as multiple photoproducts. In open ocean samples, this may require 50-701 of seawater. In coastal waters, 51 is often sufficient. In general, samples are collected by filtration. Size fraction filtration may be used to isolate particular members of the microbial community (e.g. bacterioplankton <0.8 l~m, >0.21.lm pore size). We have found Gelman SUPOR (polysulfone) filters with 142 mm diameters to work well. Filters are collected, folded, and placed in 2 oz polyethylene bags. Filters are stored at -80°C until extraction. To extract DNA remove the filter from the freezer and immediately crush the filter in the bag before it thaws. Filter pieces were then poured into a 50 ml Oak Ridge centrifuge tube. • Add 5 ml of STE (I 0 mM Tris, pH 8; I mM EDTA; I00 mM NaCI) containing 1% SDS (sodium dodecyl sulfate) to each tube. Cap, vortex for 15 s, and place in a boiling water bath for 5 rain. • Aspirate the lysate and place in a new centrifuge tube.Wash the filter pieces with an additional 5 ml of STE, vortex for 15 s, and combine with the lysate. • Extract with I 0 ml of chloroform:isoamyl alchohol (24:1). Collect the aqueous phase after centrifuging at 4°C for 30 min at 3000 rpm. • Decant the aqueous phase into a new Oak Ridge tube, add I 0 lal of a 25 mg ml ' glycogen solution, 0. I volume of 3 M sodium acetate (pH 5), and an equal volume of isopropanoI.Vortex and precipitate the sample overnight at -20°C. • Collect the precipitate by centrifugation at 12000g and 4°C for 30 rain. Decant the supernatant and wash the pellet with 10 ml cold 70% ethanol. Centrifuge at 12 O00g at 4°C for I 0 min. Decant the supernatant.
476
• Re-suspend the pellet in I ml of STE before transferring to a 2 ml microfuge tube. Re-precipitate as above, dry the pellet then re-suspend in 200 pl STE. DNA concentrations are determined spectrofluorometrically (Paul and Myers, 1982) using either a Picogreen (for native DNA) or Oligreen (for denatured DNA) reagent from Molecular Probes (Eugene, OR).We prefer the Oligreen method since it quantifies DNA concentrations immediately prior to analysis.
Competitive binding assay (RIA) The RIA is s i m p l y the basic i m m u n o p r e c i p i t a t i o n reaction outlined a b o v e into which a standard or s a m p l e D N A has been a d d e d to c o m p e t e with the radiolabeled p r o b e for a n t i b o d y binding. Hence, the p r o c e d u r e is exactly the s a m e as that used for i m m u n o p r e c i p i t a t i o n with the following additions/modifications. • A single dilution of antiserum is used. This dilution is determined from immunoprecipitation analyses of binding activity (see above) and should yield 30-60% of the radiolabeled probe in the immune pellet. • For quantification of CPDs or (6-4)PDs a dose response of heat-denatured salmon testes (Sigma) UV-irradiated DNA is used as standard (Table 23. I).We routinely use doses of 3, I 0, 30, 100, and 300 J m 2 as our standard curve and assay the same amount of standard as sample DNA.We have determined rates of photoproduct induction using independent analysis of CPDs as T4 UV endonuclease-sensitive sites and (6-4) photoproducts as photoinduced alkalilabile sites (Mitchell et al., 1990). From these values the UVC doses are converted to photoproduct frequencies using 8. I CPDs and 1.56 (6-4) photoproducts per megabase DNA per joule m 2.When relative, rather than exact, amounts of CPDs or (6-4)PDs are adequate for experimental purposes (as in DNA repair experiments) the sample harvested at the time of irradiation can be diluted in half-log increments to generate a standard. From this type of standard curve data representing percentage photoproducts remaining (or repaired) can be generated. • Unlabeled competitor mammalian DNA, radioactive ligand, and diluted antibody are incubated together for 3 h at 37°C with gentle rotation (optional). (As above, it is prudent to perform a preliminary titration of sample DNA to determine the amount required for adequate inhibition in the RIA).The total volume of sample DNA added can vary within certain limitations. Sample volumes <100 [JI do not significantly effect the reaction conditions (e.g. total binding). Sample volumes > 100 pl can be used, however, the total reaction volume should be increased accordingly (i.e. doubled).
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APPLICATIONS We h a v e used the described m e t h o d to s t u d y D N A d a m a g e in a wide variety of systems, from the Southern Ocean (Jeffrey et al., 1997) to coral reef microbial c o m m u n i t i e s (Lyons et al., 1998). Tlle protocol w o r k s
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equally well for bacterioplankton, phytoplankton (Karentz et al., 1991), and fish larvae (Malloy et al., 1997). Most often, the technique has been used to determine the amount of UVB-induced DNA damage in microbial communities (Jeffrey et al., 1996a,b). This has been done as a function of time of day, depth in the water column, cell size fraction, or seasonal comparisons. It is also possible to examine rates of DNA damage repair by incorporating broadband spectral filters (e.g. Mylar 500 D or acrylics, Aas et al., 1996) post-UVB irradiation. The main obstacle to doing this with marine microbial communities is the large volume required to be filtered for each time point. We have had some success by constructing UV-transparent incubators that will hold up to 200 1of seawater at ambient temperatures from which samples may be collected over time. High cell densities in laboratory cultures allow more detailed experimentation to be designed (Joux et al., 1999). The DNA damage assays are identical, with only minor modifications in the DNA extraction protocol required. The kinetics of DNA damage induction and repair may be determined as a function of dose response, for instance, and comparisons made between different strains. Smaller volumes may allow greater spectral resolution of a UV response by incorporating additional optical filters (e.g. Schott filters, Schott Glass Technologies, Duryea, PA) which are not available in sizes needed to incubate seawater samples.
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DATA ANALYSIS A sample Excel spreadsheet for quantification of CPDs or (6-4)PDs is shown in Table 23.1. A sample Excel spreadsheet for quantification of relative photoproducts remaining at specific times post UV-irradiation (e.g. in a DNA repair experiment) is shown in Table 23.2.
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CONCLUSIONS
AND FUTURE DIRECTIONS
Different types of DNA damage have different immunogenicities and may not be suitable for the production of antibodies in rabbits or mice. We have been unsuccessful in raising rabbit antisera against DNA irradiated with UVA light, DNA cross-linked with mitomycin C, or DNA homopolymers or alternating copolymers irradiated with UVC light. These failures may be due to the low antigenicity of the damage itself (e.g. UVA, mitomycin C), immunogen stability in the host (e.g. digestion of oligonucleotides or polynucleotides), or host variability (i.e. not enough rabbits). We have been successful, however, at raising polyclonal antibodies against UVC light, triplet-sensitized UVB light, DNA containing acetylaminofluorene adducts, DNA containing benzo[a]pyrene diolepoxide adducts, DNA treated with osmium tetroxide (i.e. thymine glycols), and HPLC-purified 8-oxodeoxyguanosine covalently linked to a protein hapten. 480
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RIA and ELISA are p o w e r f u l techniques for the sensitive and specific detection of genotoxic d a m a g e in DNA. Other techniques, such as quantitative i m m u n o c y t o c h e m i s t r y , immunohistochemistry, and i m m u n o e l e c tron m i c r o s c o p y have been designed to detect D N A d a m a g e in situ, thus visualizing the distribution of d a m a g e in tissues and cells. D a m a g e d D N A m a y be separated from u n d a m a g e d D N A fragments b y i m m u n o precipitation, in-tmunoaffinity chromatography, or nitrocellulose binding. Enrichment of d a m a g e d D N A f r a g m e n t s using antibodies has been c o m b i n e d with PCR amplification and Southern analysis to determine the genomic distribution of D N A d a m a g e (Hochleitner et al., 1991) and with HPLC (or ligation-mediated PCR) to increase the resolution and, hence, sensitivity of detection ( G r o o p m a n et al., 1992).
References Aas, P., Lyons, M., Pledger, R., Mitchell, D. L. and Jeffrey, W. H. (1996). Inhibition of bacterial activities by solar radiation in nearshore waters and the Gulf of Mexico. Aquatic Microbial Ecol. 11, 229-238. Cadet, J. and Vigny, P. (1990). The photochemistry of nucleic acids. In: Biooor~aJTic PhotochenlistJ 7, Vol. 1: Ptlotochenlistry and the Nuch'ic Acids (H. Morrison, Ed.), pp. 1-272. John Wiley and Sons, New Yurk. Calkins, J. (Ed.) (1982). The Roh' qf Solar Llltravi~det RadiatioH iH Marine Ecosystems. Plenum Press, New York. Chin-Leo, G. and Kirchman, D. L. (1988). Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine. Appl. Euviron. Micmbiol. 54, 1934 1939. Coffin, R. B., Velinsky, D., Devereux, R., Price, W. A. and Cifuentes, L. (1990). Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria. Appl. Euvirol~. Micmbiol. 56, 2012-2020. Cota, G. E, Kottmeier, S. T., Robinson, D. H., Smith, W. O. and Sullivan, C. W. (1990). Bacterioplankton in the marginal ice zone of the Weddel] Sea: biomass, production and metabolic activities during Austral summer. Deep-Sea Res. 37, 1145-1167. Falkowski, P. G. and Woodham, A. D. (1992). Prima12t/Production aud Biogeochemical Cycles in the Sea. Plenum Press, New York. Fuhrman, J. A. and Azam, E (1980). Bacterioplankton secondary production estimates for coastal waters of British Columbia, Antarctica, and California. App[. El~virou. Microbiol. 39, 1085 1095. Fuhrman, J. A. and Azam, E (1982). Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: evaluation and field results. Mar. Biol. 66, 109-120. Groopman, J. D., Zhu, J. Q., Donahue, P. R., Pikul, A., Zhang, L. S., Chen, J. S. and Wogan, G.N. (1992). Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region, People's Republic of China. Ca~Tcer Res. 52, 45-52. Hansen, R. B., Sharer, D., Ryan, T., Pope, D. and Lowery, H. K. (1983). Bacterioplankton in the Antarctic ocean waters during late Austral winter, abundance, frequency of dividing cells, and estimates of production. Appl. EllviroJl. Microbiol. 45, 1622 1632. Herndl, G. ]., M/_iller-Niklas, G. and Frick, J. (1993). Major role of ultraviolet-B in controlling bacterioplankton growth in the surface layer of the ocean. Nature 361, 717-719.
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Hochleitner, K., Thomale, J., Nikitin, A. Yu and Rajewsky, M. E (1991). Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences. Nucleic Acids Rcs. 19, 4467-4472. Jeffrey, W. H., Aas, P, Lyons, M. M., Pledger, R., Mitchell, D. L. and Coffin, R. B. (1996a). Ambient solar radiation induced photodamage in marine bacterioplankton. Photochenl. Photobiol. 64, 419-427. Jeffrey, W. 1t., Miller, R. V. and Mitchell, D. L. (1997). Detection of ultraviolet radiation induced DNA damage in microbial communities of the Gerlache Strait. Aittarctic J. LIS 32, 85-87. Jeffre}; W. H., Pledger, R. J., Aas, P., Hager, S., Coffin, R. B., Von Haven, R. and Mitchell, D. L. (1996b). Diel and depth profiles of DNA photodamage in bacteriuplankton exposed to ambient solar radiation. Mar'. Ecol. Prog. Ser. 137, 283-291. Joux, E, Jeffrey, W. H., Lebaron, I~ and Mitchell, D. (1999). Marine bacteria display diverse responses to u/traviolet-B radiation. Appl. Environ7. Microbiol. 65, 3820-3827. Karentz, D., Cleaver, J. E. and Mitchell, D. L. (1991). Cell survival characteristics and molecular responses of antarctic phytoplankton to ultraviolet-B radiation. J. Phycol. 27, 326-341. Lamola, A. A. and Yamane, T. (1967). Sensitized photodimerization of thymine in DNA. Proc. Natl. AcmL Sci. LISA 58, 443-446. Lyons, M. M., Aas, P., Pakulski, J. D., Van Waasbergen, L., Mitchell, D. L., Miller, R. V. and Jeffrey, W. H. (1998). Ultraviolet radiation induced DNA damage in coral reef microbial communities. Mar. Biol. 130, 537-543. Malloy, K. D., Holman, M. A., Mitchell, D. and Detrich, H. W. III (1997). Solar UVBinduced DNA damage and photoenzymatic repair in Antarctic zooplankton. Proc. N~ttl. Acad. Sci. USA 94, 1258-1263. Mitchell, D. L. (1995). Ultraviolet radiation damage to DNA. In: Molecular Biology amt Bioteclmology: A Comptvhensive Desk Reference (R. A. Meyers, Ed.), pp. 939-943. VCH Publishers, New York. Mitchell, D. L. (1996). Radioimmunoassay of DNA damaged by ultraviolet light. In: Techllologics (or Detectiol~ qf DNA Damage ~TmtMutatiotzs (G. Pfeifer, Ed.), pp. 73-85. Plenum, New York, Mitchell, D. L. and Clarkson, J. M. (1981). The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA. Biochim. Biophys. Acta 655, 54 60. Mitchell, D. L. and Nairn, R. S. (1989). The biology of the (6-4) photoproduct. Photochem. Photobiol. 49, 805-819. Mitchell, D. L., Brash, D. E. and Nairn, R. S. (1990). Rapid repair of pyrimidine (6-4)pyrimidone photoproducts in human cells does not result from change in epitope conformation. NHcleic Acids Res. 18, 963-971. Paul, J. H. and Carlson, D. (1984). Genetic material in the marine environment: implication for bacterial DNA. Limm~l. Oceam~gr. 29, 1091-1097. Paul, J. H. and Myers, B. (1982). Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258. Appl. E1~virott. Microbiol. 43, 1393-1399. Paul, J. H., Jeffrey, W. H. and Deflaun, M. E (1985). Particulate DNA in subtropical oceanic and estuarine planktonic environments. Mar. Biol. 90, 95-101; Appl. EHvirotz, Microbio[. 43, 1393-1399. Rosenstein, B. S. (1984). Photoreactivation of ICR 2A frog cells exposed to solar UV wavelengths. Photochcm. Photobiol. 40, 207-213. Simon, M. and Azam, E (1989). Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Pro~,,. Set. 51,201-213.
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Sullivan, C. W., Cota, G. E, Krempin, D. W. and Smith, W. O. Jr. (1990). Distribution and activity of bacterioplankton in the marginal ice zone of the Weddell-Scotia Sea during austral spring. Mar. Ecol. Prog. Set. 63, 239-252. Vincent, W. E and Roy, S. (1993). Solar ultraviolet-B radiation and aquatic primary production: damage, protection, and recovery. Environ. Rev. 1, 1-12. Worrest, R., Dyke, H. V. and Thomson, B. (1978). Impact of enhanced simulated solar ultraviolet radiation upon a marine community. Photochem. Photobiol. 27, 471-478. Worrest, R., Wolniakowski, K., Scott, J., Brooker, D. and Dyke, H. V. (1981). Sensitivity of marine phytoplankton to UV-B radiation: impact upon a model ecosystem. Photochem. Photobiol. 33, 223-227. Zepp, R., Callaghan, T. and Erickson, D. (1995). Effects of increased solar ultraviolet radiation on biogeochemical cycles. Ambio 24, 181-187.
List of suppliers Amersham Pharmacia Biotech, Inc. 800 Centennial Avemte P.O. Box 1327 Piscataway, NJ 08855-1327, USA Tel: 732-457-8000 Fax: 732-457-0557 Radio-labeled dNTPs; Tissue solubilizer
Eastman Kodak Company Kodacel Marketing Roll Coating Division 1669 Lake Avenue Kodak Park, Building 21, Floor 2 Rochestel; NY 14652-3202, USA Teh 800-367-5235 Fax: 716-477-3465 Kodacel
Boehringer-Mannheim E Hoffmann-La Roche Ltd CH-4070 Basel Switzerland Teh ++41 / 61 688 11 11 Fax: ++41 / 61 691 93 91
Fisher Scientific 585 Alpha Drive Pittsburgh, PA 15238, USA Tel: 800-766-7000
Nick Translation kit; DNase-free RNAse A; Tris saturated phenol
Gelman Sciences (Pall Gelman Sciences) 600 S. Wagner Road Ann Arbot, M148103, USA Tel: 800-521-1360 Fax: 800-913-6440
Liquid scintillation cocktail
Calbiochem-Novabiochem Corporation 10394 Pacific Center Court San Diego, California 92121, USA Mailing Address: P.O. Box 12087 La Jolla, California 92039-2087, USA Teh 858- 450-9600 Fax: 858- 453-3552 N o r m a l rabbit sera; Goat antirabbit (IgG)
484
Supor filters
International Light, Inc. 17 Graf Road Newbu ryport, MA 01950, USA Tel: 978-465-5923 Fax: 978-462-0759 UV r a d i o m e t e r
Pharmacia and Upjohn 100 Route 206 North Peapack, New Jersey 07977, USA Tel: 908-901-8000; 888-768-5501 Fax: 908-901-8379
Millipore Corp. 186 Middlesex Turnpike Burlington, MA 01803, USA Tel: 781-533-6000
HPLC water
Nick Column Molecular Probes PO Box 22010 Eugene, OR 97402-0469, USA 4849 Pitchford Avenue Eugene, OR 97402-9165, USA Teh 541- 465-8300 Fax: 541-344-6504
Sigma Chemical 3050 Spruce Street St. Louis, MO 63103, USA Tel: 314-771-5765
DNA; methylated BSA; gelatin
Oligogreen nucleic acid dye New England Nuclear (NEN) NEN® Life Science Products, Inc. 549 Albany Street Boston, MA 02118-2512, USA Teh 800-551-2121 or 617-482-9595 Fax: 617-482-1380
Radiolabeled dNTPs
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