EMÏÏT3TÏÏMWÏÏ0 Jean Cadet CEA/Département de Recherche Fondamentale sur la Matière Condensée SESAM/LAN, BP 85X F-38041 Grenoble, France
Michael Weinfeld Department of Radiobiology Cross Cancer Institute Edmonton, Alberta T6G 1Z2, Canada
The purine and pyrimidine bases in DNA are critical cellular targets of UV l i g h t a n d v a r i o u s oxidizing agents and processes, including ionizing radiation, photoexcited dyes, antibiotics, asbestos, and oxidative metabolism. As a result, DNA photoproducts such as cyclobutadipyrimidines and pyrimidine(6-4)pyrimidone p h o t o a d d u c t s , a n d oxidized DNA lesions such as 5,6-dihydroxy5,6-dihydrothymine (thymine glycol), have been implicated in cell lethality, mutagenesis, and carcinogenesis (1, 2). There is, therefore, increasing interest in the development of accurate assays for measuring DNA base 0003-2700/93/0365-675A/$04.00/0 © 1993 American Chemical Society
damage. Such assays should help to define the biological consequences of photo-induced a n d oxidative DNA modifications and to identify enzymatic repair pathways. For most cases, the detection and quantitation of a specific DNA base modification within either isolated
REPORT cells or whole organs present a challenging analytical problem. In this REPORT we describe recent methodological aspects of the measurement of DNA damage, with emphasis on chromatographic and electrophoretic assays that allow quantitation of individual photo-induced and oxidative DNA base modifications. The bulk of the assays are designed for monitoring the formation of known, targeted base damage within DNA.
General approaches A major limitation of these assays is their sensitivity, which must be high enough to allow the measurement of
at least one modification in 1 0 4 - 1 0 6 n o r m a l b a s e s w i t h i n a few micrograms of DNA. Two main approaches have been developed. In the first, the DNA is kept intact and lesions are m e a s u r e d either by immunological methods or by the nicking activity of DNA repair enzymes in association with sedimentation and gel-sequencing techniques t h a t quantify the number of strand breaks. The second approach requires eit h e r q u a n t i t a t i v e acidic hydrolysis (base release) or enzymatic digestion (release of nucleosides, nucleotides, or short oligonucleotides). The comp o u n d s of i n t e r e s t a r e s e p a r a t e d from the overwhelming majority of normal DNA constituents, and the complex mixture of DNA lesions is analyzed. Separation can be achieved by using HPLC, GC, and capillary electrop h o r e s i s (CE), a n d t h e s e p a r a t e d DNA lesions can be detected on line by various sensitive and sometimes specific methods such as MS, fluorescence, amperometry, or radioactivity detection. Some measurements require prederivatization, postlabel-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993 · 675 A
REPORT ing, or postcolumn reaction of the samples (Table I). HPLC
Modified DNA components obtained by either mild acidic hydrolysis or enzymatic digestion of DNA usually are efficiently separated by HPLC on octadecylsilyl silica gel (ODS) col u m n s . Electrochemistry (EC), MS, and fluorescence are normally used for monitoring the formation of oxi dized and photo-induced DNA dam age. In addition, an HPLC-radioactivity method has been developed to quantify far-UV and solar radiationinduced cyclobutane pyrimidine dimers in m a m m a l i a n cells (1, 3). This assay requires radioactive prelabeling of thymine bases within cel lular DNA, an approach t h a t is not s u i t a b l e for m o n i t o r i n g oxidative DNA d a m a g e because of t h e selfradiolysis processes that occur. On-line EC detection coupled with HPLC separation represents a sensi tive a n d a c c u r a t e m e t h o d for t h e m e a s u r e m e n t of readily oxidizable compounds. It has been successfully applied to the specific determination of four oxidized purine nucleobases and nucleosides with low redox po tentials : 7,8 - dihydro - 8 - oxoadenine, 7,8-dihydro-8-oxoguanine, and their corresponding 2'-deoxyribonucleosides (2, 4). This technique was recently used for the determination of oxidized py rimidine components, including 5-hydroxyuracil, 5-hydroxycytosine, and related nucleosides (5). The limit of detection for these compounds is close to one modification in 10 5 nor mal bases, an amount sufficient for application of the assay to the mea surement of the oxidized bases and nucleosides, especially 7,8-dihydro 8-oxo-2'-deoxyguanosine in cellular DNA a n d in biological fluids (2). Note, however, t h a t this assay r e quires at least 25 μg of DNA. Thermospray MS coupled with on line HPLC is suitable for the analy sis of various radiation-induced de composition products of thymidine, including N-(2-deoxy-$-D-erythropentofuranosyDformamide, the four cis a n d t r a n s d i a s t e r e o i s o m e r s of 5,6 - dihydroxy - 5,6 - d i h y d r o t h y m i dine, and the 5R and 5S forms of 5,6dihydrothymidine, as shown in Fig ure 1. Recording only a particularly abundant and product-specific ion in the selected ion m o n i t o r i n g (SIM) mode allows a limit of detection of the two modified 2'-deoxyribonucleos i d e s in t h e low-picomole r a n g e , which is equivalent to approximately one lesion per 10 5 normal bases in
Table 1. Methodologies for measuring photo •induced and oxidative DNA damage Methods H PLC/electrochemistry HPLC/MS (thermospray) HPLC/fluorescence GC/SIM-MS GC/ECNI-MS HPLC (electrophoresis/ enzymatic 3έ Ρ-postlabeling) HPLC/chemical 3 H-postlabeling HPLC/postfluorescence Immunology (RIA/ELISA assays) UV endonuclease/gel electrophoresis/ fluorescence Endonuclease III— FPG protein/ alkaline elution assay a
DNA
Sensitivity*
Amount of DNA (μβ)
Reference
Hydrolyzed Hydrolyzed Hydrolyzed Hydrolyzed Hydrolyzed Hydrolyzed
1 x10~ 5 1 x10~ s 5x10-5 1 x10"5 2x10~ 4 1 x10~ e
25-50 30-60 4-8 50-100 10-50 1-5
2,4,5 6 8 10-13 14,15 19-23
Hydrolyzed
0.5-3x10-"
100
24
1 x1010- 5 -10" e
100 2-10
25 27-30
Intact
1 x10-6
30-50 ng
33,34
Intact
5x10"7
10
37
Hydrolyzed Intact
6
Sensitivity is indicated with respect to normal bases.
Figure 1. HPLC/thermospray MS of the main radical oxidation products of thymidine. Total ion chromatogram (TIC) profile obtained on an ODS column with 0.1 M ammonium acetate (pH 6) as the mobile phase.
30 \ig of DNA (6). Another interesting application of t h e r m o s p r a y LC/MS with SIM in volves m e a s u r i n g the predominant malonaldehyde-guanine adduct with a threshold of detection close to 250 fmol (7). As with most GC/MS a s says, accurate quantitative measure ment requires internal calibration by adding a known amount of the stable isotope standards prior to the HPLC separation. In addition to sensitivity, t h e m a i n a d v a n t a g e of the LC/MS assays is the possibility of acquiring
676 A . ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
accurate structural information. Other promising developments in volve techniques such as continuous fast atom bombardment (FAB), elec trospray, and atmospheric pressure ionization MS, which can be used on line with microanalytical HPLC and CE separations. Micellar electrokinetic capillary chromatography com bined with electrospray MS appears to be particularly appropriate for the m e a s u r e m e n t of modified dinucleoside monophosphates that are resis t a n t to enzymatic digestion [e.g., cy-
clobutadipyrimidines and (6-4) photoproducts]. F l u o r e s c e n c e d e t e c t i o n on l i n e with HPLC constitutes a sensitive approach for monitoring the forma tion of DNA photoproducts. One in teresting application of this assay is the detection of [2+2] photocycloaddition products arising from the r e a c t i o n of p h o t o e x c i t e d f u r o coumarins through their furan moi ety with thymidine (1). In addition, a method was recently developed for the determination of the main DNA photolesions induced by the monofunctional 3-carbethoxypsoralen (3-CPs) in yeast and mam malian cells upon exposure to UVA radiation (8). This assay is based on the enzymatic digestion of the ex tracted DNA from i r r a d i a t e d cells, followed by r e v e r s e d - p h a s e HPLC separation coupled on line to a highly sensitive fluorescence detector. Detection and quantitative measure ment of the two cis-syn diastereoisomers of furan-side 3-CPs monoadducts to thymidine ( 3 - C P s o d T h d ) at a level as low as 1.3 and 0.7 per 10 4 base pairs, respectively, are pos sible, as shown in Figure 2. The assay, which requires neither prelabeling of the cells nor the use of radioactive psoralen derivative, is suitable for studying the individual r e p a i r kinetics of each of t h e two 3 - C P s o d T h d diastereoisomers. This approach could also be used to mea s u r e a wide r a n g e of p y r i m i d i n e
0
10
20
Retention time (min)
o f u r a n - s i d e monoadducts of psor alens, compounds t h a t are used in cosmetology and photochemotherapy of various skin diseases. A second important application of the HPLC-fluorescence assay is the measurement of pyrimidine (6-4) pyrimidone photoproducts, which rep r e s e n t a m a j o r c l a s s of f a r - U V induced DNA p h o t o l e s i o n s (1, 3). Mild hydrolysis of irradiated DNA by hydrogen fluoride stabilized with py ridine quantitatively induces the re lease of three base photoadducts, in cluding thymine-cytosine, t h y m i n e thymine, and cytosine-thymine (6-4) photoadducts (3). The released DNA lesions are efficiently s e p a rated by HPLC and q u a n t i t a t e d by on-line fluorescence detection. The formation of the t h y m i n e - t h y m i n e (6-4) photoadduct, which h a s a q u a n t u m yield of f l u o r e s c e n c e of 0.21, can be detected in the femtomole range. Significant enhancement in t h e s e n s i t i v i t y of d e t e c t i o n of these two classes of DNA photoprod ucts is expected from the use of syn chronous laser-induced fluorescence detectors. Pyrimidine hydroperoxides can be selectively detected by a postcolumn reaction because they are able to ox idize the ferrous sulfate-xylenol or ange reagent. The resulting oxidized complex exhibits an intense absorp tion around 540 nm. This assay has been used for on-line colorimetric m e a s u r e m e n t of five m e n a d i o n e -
U2(^=H)J2(RV=CH$
Figure 2. HPLC with fluorescence detection of the cis-syn diastereoisomers of the furan-side 3-carbethoxypsoralen monoadducts to thymidine (T17 T2) and 2'-deoxyuridine (U,, U2) with dR = 2-deoxy-p-D-eryinro-pentofuranosyl. ODS column (25 cm χ 0.46 cm i.d.) with methanol/water as the eluent and a flow rate of 1 mLVmin. Detection: excitation, 357 nm; emission. 415 nm.
photosensitized formation products of t h y m i d i n e hydroperoxides after their complete separation on an ODS column ( F i g u r e 3). The d e t e c t i o n limit for each of the thymidine hy droperoxides, including 5-hydroperoxymethyl-2'-deoxyuridine and the four cis and t r a n s diastereoisomers of 5 - h y d r o p e r o x y - 6 - h y d r o x y - 5 , 6 dihydrothymidine, is 10 pmol (9). GC/MS Two approaches, G C / S I M - M S and G C / e l e c t r o n c a p t u r e n e g a t i v e ion (ECNI) MS, are commonly used. The combination of on-line MS with the high resolving power of capillary GC has already been used, primarily by Dizdaroglu's group (10), in monitoring the formation of a wide range of ra diation-induced and oxidative DNA base damage. This technique re quires the derivatization of the mod ified bases or nucleosides, which are obtained by either acidic hydrolysis or enzymatic digestion of extracted DNA. It was recently shown that the detection of ierf-butyldimethylsilyl derivatives of modified nucleobases, including 5 - h y d r o x y m e t h y l u r a c i l , 5-formyluracil, 5-hydroxycytosine, and 5-hydroxyuracil, has a sensitiv ity about fourfold that of related trimethylsilylated compounds (11). Under these conditions, using SIM m a k e s it possible to d e t e c t 5 - h y droxymethyluracil with a detection limit of 20 pg. This is about 50-fold less sensitive t h a n the HPLC assay with 32 P-postlabeling, but GC/MS is m o r e s u i t e d to r o u t i n e a n a l y s e s . G C / M S a n a l y s i s , a t l e a s t for t h e above compounds, is as sensitive as t h e H P L C - E C a s s a y s , a n d it pro vides structural information. How ever, when SIM is used, at least two ions must be present in the correct abundance ratio and must coelute at the proper retention time in order to obtain accurate identification and quantitation. Several oxidative DNA a l t e r a t i o n s can be measured accu r a t e l y by u s i n g t h e stable isotope dilution technique because many tri or tetra-isotopically enriched modi fied bases and nucleosides are avail able (11-13). The second approach, GC/ECNIMS, requires off-line alkylation of modified bases to their highly elec t r o p h o n e pentafluorobenzyl (PBF) derivatives after initial isolation by r e v e r s e d - p h a s e HPLC. Use of this technique allowed efficient and sen sitive determination of cyclobutadipyrimidines; limits of detection of the lesions were in t h e femtomole range (14). Another interesting application of
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REPORT this sensitive approach is the mea surement of 5-hydroxymethyluracil, a radical oxidation product of t h y mine, within DNA (15). Low femtomole-level detection of the oxidized base as its t r i - P B F derivative was achieved by u s i n g either capillary GC or a moving-belt LC interface. Advantages and limitations of HPLC and GC/MS assays are critically r e viewed in a recent comprehensive survey (16). 32
P postlabeling assays
Randerath and co-workers (17) de veloped these methods to m e a s u r e carcinogen-DNA adducts. Damaged DNA is first digested enzymatically to nucleoside 3'-monophosphates or very short oligonucleotides (depend ing on the enzymes used for diges tion and the damage to DNA) t h a t are then enzymatically radiolabeled by incubation with 3 2 P-labeled ATP and phage T4 polynucleotide kinase (Figure 4). A variety of techniques, including two-dimensional TLC and HPLC, can be used to analyze t h e radiolabeled products. These p o s t l a b e l i n g a s s a y s h a v e two important advantages over prelabeling methods. First, because there is no requirement for prelabeling DNA, the assay is particularly useful for the study of DNA damage in biological samples. Second, be cause 3 2 P - l a b e l e d ATP is commer cially available at high specific activ ity (> 3000 Ci/mmol), t h e assay is extremely sensitive. For example, using ATP with a specific activity of 3000 C i / m m o l , 1 fmol of l a b e l e d damaged DNA product r e p r e s e n t s 6000 dpm. In the original approach (17) dam aged DNA was digested by micrococcal nuclease and calf spleen p h o s phodiesterase. This combination of enzymes hydrolyzes DNA to nucleo side 3'-monophosphates. However, because the normal and the damaged bases are released as m o n o n u c l e otides, t h e r e is a high background from undamaged DNA. This problem can be circumvented in two ways. The first method in volves chromatographic enrichment of the modified nucleoside 3'-mono phosphates prior to labeling. For ex ample, 5,6-dihydroxy-5,6-dihydrothymidine 3'-monophosphate can be enriched by affinity chromatogra phy on a p h e n y l b o r o n a t e column, t a k i n g a d v a n t a g e of t h e cis diol structure of the modified base (18). The second approach is to selectively dephosphorylate the unmodified nucleoside 3 ' - m o n o p h o s p h a t e s by nuclease P I prior to labeling. (Nucle
Figure 3. Postcolumn reaction HPLC analysis of the five pyrimidine hydroperoxides (dR = 2-deoxy-3-D-eryi/7ro-pentofuranosyl) obtained by menadione photooxidation of thymidine. The separation is achieved on an analytical (25 cm χ 0.46 cm i.d.) Ultrasphere column with water (pH 6) as the mobile phase.
osides are not substrates for polynu cleotide kinase and therefore will not be labeled.) This procedure is highly dependent on substrate recognition by nuclease P I and is not applicable to all damage. More r e c e n t l y , it h a s b e e n o b served t h a t certain modified bases inhibit DNase 1 and phosphodi esterase 1 (snake venom phosphodi e s t e r a s e ) from cleaving t h e p h o s phodiester linkage immediately 5' to the site of damage. As a result, when d a m a g e d DNA is i n c u b a t e d w i t h these enzymes and with alkaline p h o s p h a t a s e , t h e l e s i o n s a r e ob tained in the form of either dinucleo side monophosphates or—in the case of cyclobutadipyrimidines and (6-4) photoproducts — trinucleoside di p h o s p h a t e s (Figure 4). Again, u n damaged DNA is reduced to nucleo sides and hence does not contribute to background. Following radioactive phosphorylation, the labeled oligonu cleotides are examined by a combi nation of polyacrylamide gel electro phoresis and HPLC (19). These two basic protocols are, to some extent, complementary because there are certain DNA modifications for which each protocol is superior. For example, photo-induced cyclobu tadipyrimidines and oxidized sugar residues, such as the phosphoglycolate groups produced in DNA by ion izing radiation and bleomycin, can only be d e t e c t e d by t h e selective
678 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
phosphorylation protocol. (The prod u c t s of d i g e s t i o n by micrococcal nuclease and calf spleen phosphodi esterase containing these modifica tions are not substrates for polynu cleotide kinase.) However, bases with minor alterations, such as 7,8-dihydro-8-oxoguanine and 5-hydroxyme thyluracil, can only be detected by the Randerath approach, because these a l t e r a t i o n s do not i m p e d e s n a k e venom phosphodiesterase. To date, most published r e p o r t s regarding the use of 3 2 P postlabeling assays for quantifying oxidative and p h o t o - i n d u c e d DNA d a m a g e h a v e dealt with method development. However, there are several studies in which the assay was used to assess oxidative base damage in biological systems. For example, it h a s been shown that hydrogen peroxide treat ment of the bacterium Proteus mirabi lis dramatically enhances the yield of a d e n i n e N-oxide in t h e b a c t e r i a l DNA (20). This assay has also proven c o n v e n i e n t for t h e s t u d y of enzy matic repair of certain lesions, espe cially those for which the chemical s y n t h e s i s of defined s u b s t r a t e s is complicated (21). More exhaustive r e v i e w s of t h e a p p l i c a t i o n of 3 2 P postlabeling assays to oxidative DNA damage have been published (22, 23). Chemical postlabeling assays Two chemical postlabeling assays of nucleosides and nucleotides obtained
by enzymatic digestion of oxidized DNA have recently been developed. Both approaches involve HPLC prepurification of the compounds of in terest, which in a subsequent step are chemically modified. One a p proach involves acetylation of the hydroxyl groups of several oxidized nucleosides, including the (5R, 6S)and (5S, 6i?)-diastereoisomers of 5,6dihydroxy - 5,6 - d i h y d r o t h y m i d i n e , 5 -hydroxymethyl - 2'- deoxyuridine, 5-formyl-2'-deoxyuridine, and 7,8dihydro- 8 -oxo- 2'- deoxyguanosine by [ 3 H]-acetic anhydride (24). The re sulting more hydrophobic 0- acetyl ated nucleosides are further sepa rated by HPLC on an ODS column using authentic cold markers as in ternal standards. This assay, which allows the detection of 7,8-dihydro8-oxo-2'-deoxyguanosine at a level as low as 2.5 pmol, has been applied to t h e m e a s u r e m e n t of o x i d a t i v e DNA damage in the organs of r a t s exposed to the tumor promoter 12-0tetradecanoylphorbol 13-acetate. A second method, the fluorophore l a b e l i n g of o x i d i z e d n u c l e o s i d e 5'-monophosphates, can be achieved through phosphoramidate intermedi
ates by reaction with dansyl chloride and fluorescein isothiocyanate (25). The sensitivity of detection of the dansylphosphoramidate derivatives of 5,6-dihydroxy-5,6-dihydrothymi dine and 7,8-dihydro-8-oxo-2'deoxyguanosine determined by on line fluorescence m e a s u r e m e n t is one modification per 10 6 normal nu cleosides in a sample size of 100 μg of DNA. These techniques have been recently reviewed (23). Immunochemical detection Immunological detection h a s long b e e n r e c o g n i z e d as a p o t e n t i a l l y powerful tool in the analysis of genot o x i n - m o d i f i e d DNA. As e a r l y as 1966, Levine a n d co-workers p r e p a r e d a n t i b o d i e s a g a i n s t far-UVinduced photoproducts of DNA (26). Since then, antibodies have been de rived against several oxidative and photo-induced DNA lesions, includ ing 5,6-dihydroxy-5,6-dihydrothymine, 7,8-dihydro-8-oxoadenine, cyc l o b u t a n e - p y r i m i d i n e dimers, and (6-4) photoproducts (1, 3). The main advantages of immunochemical de tection are specificity (especially if monoclonal a n t i b o d i e s are u s e d ) ,
Figure 4. Experimental procedure for 32 P postlabeling analysis of modified dinucleoside monophosphates (left) and nucleoside 3'-monophosphates (right).
sensitivity (subfemtomole level), and simplicity once antibodies have been generated. Like other haptens, modified bases must be linked to macromolecules to elicit an immune response. The prin cipal methods employed in the prep aration of immunogens are the syn t h e s i s of modified n u c l e o s i d e s or nucleotides that are then covalently coupled to proteins such as bovine s e r u m a l b u m i n or keyhole limpet haemocyanin, and the modification of single- or double-stranded DNA, which is then electrostatically bound to m e t h y l a t e d bovine serum albu min. In the first method, the osidic moi ety of ribonucleosides can be cleaved by periodate oxidation of the cis-diol function. The molecule then is bound to protein through the resulting aldehydic f u n c t i o n s . A l t e r n a t i v e l y , modified mononucleotides can be di rectly coupled to protein by carbodiimide-mediated condensation of the p h o s p h a t e of t h e n u c l e o t i d e w i t h protein amino groups. Problems of cross-reactivity have occurred when damaged polymeric DNA has been used as an immunogen. This became evident w h e n a polyclonal antiserum raised against U V - i r r a d i a t e d DNA recognized all the thymine- and cyto sine-contain ing cyclobutane dimers together with (6-4) photoproducts, but with a dif ferent affinity for each major class of damage (27). Thus, if damaged DNA is to be used as the immunogen, it is important to choose conditions t h a t greatly favor the production of a sin gle type of damage over any other. An example is the use of osmium tetroxide, which produces cis t h y mine glycol almost exclusively, in stead of ionizing r a d i a t i o n , which gives rise to many different chemical modifications. Similarly, the ratio of cyclobutadipyrimidines to other pho toproducts can be enhanced by acet o p h e n o n e p h o t o s e n s i t i z a t i o n of DNA when t h e DNA is exposed to 313 nm radiation (1). The antibodies raised against such DNA damage have been exploited in a variety of immunodetection meth ods. The most widely used technique is t h e w e l l - k n o w n e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y (ELISA), in which the primary antibodies, when bound to the antigen, are detected by s e c o n d a r y a n t i b o d i e s l i n k e d co valently to an enzyme such as alka line phosphatase or peroxidase. Sub s t r a t e s for t h e e n z y m e s a r e t h e n employed t h a t yield products with intense absorption at visible wave lengths. Other substrates have used
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993 · 679 A
REPORT either primary antibodies coupled to radioactive isotope (radioimmunoas say) or secondary antibodies conju gated to fluorescent compounds, such as fluorescein isothiocyanate (FITC). Immunodetection has been partic ularly effective for studying the in duction and r e p a i r of UV-induced p h o t o p r o d u c t s . N i k a i d o a n d co workers (28, 29) established mono clonal antibodies recognizing cyclobutadipyrimidines and (6-4) photoproducts. Using autoradiogra phy to detect t r i t i a t e d antibodies, they were able to observe (6-4) pho toproducts in human cells exposed to as little as 10 J / m 2 of 254-nm light. In contrast to normal proficient hu man cells, which removed more than 80% of their initial damage within 4 h of post-irradiation, cells derived from a patient with xeroderma pig mentosum (a genetic disorder char acterized by a marked sensitivity to UV light) showed almost no repair within 8 h (29). With an ELISA pro tocol, the same group demonstrated that cyclobutadithymines are excised from DNA in irradiated (40 J / m 2 of 2 5 4 - n m l i g h t ) h u m a n cells m o r e slowly t h a n (6-4) photoproducts; r e moval of t h e l a t t e r w a s v i r t u a l l y completed within 12 h of post-irradi ation, whereas at 24 h half of the cyclobutadipyrimidines still remain. Lesko and co-workers (30) were able to observe the formation of cy clobutadithymines in individual cells by FITC-labeled secondary antibod ies and computer-assisted microfluo rometry. They assessed the number of dimers in cultured Syrian hamster cells exposed to 10 J / m 2 of 254-nm light at 8 χ 10 5 per nucleus. An a l t e r n a t i v e immunodetection method for examining damage to cel lular DNA induced by ionizing radia tion, based on a monoclonal antibody to s i n g l e - s t r a n d e d DNA, h a s been developed by van der Schans and co workers (31). Under alkaline condi tions, certain types of base damage give rise to strand breaks, in addi tion to those initially induced by the radiation. At the same time, alkali treatment begins to unwind the DNA a t s t r a n d b r e a k s . T h e r e f o r e , by cleaving a n d u n w i n d i n g t h e DNA u n d e r defined conditions and t h e n using the anti-single-stranded DNA antibody in a n ELISA assay, it is possible to detect strand breaks and alkali-sensitive base damage at clin ically relevant doses of ionizing radi ation (< 2 Gy) in as few as 10 6 cells.
that cleave DNA at a phosphodiester bond adjacent to a d a m a g e d base. Modified b a s e s can, t h e r e f o r e , be quantified by i n c u b a t i n g damaged DNA with such an enzyme and then using one of several approaches to d e t e r m i n e t h e n u m b e r of s i n g l e strand breaks. Enzymes t h a t have been used in this fashion include Micrococcus luteus UV endonuclease V a n d T4 phage endonuclease, both of which specifi cally recognize c y c l o b u t a d i p y r i m idines; Escherichia coli endonuclease III, which incises DNA at a variety of oxidized pyrimidine modifications such as thymine glycols; the forma m i d o p y r i m i d i n e - D N A glycosylase (FPG) protein, which is able to excise 7,8-dihydro-8-oxoguanine and formamidopyrimidines; the UVR ABC exinuclease complex, which recog nizes a variety of bulky DNA photod a m a g e such as cyclobutadipyrimidines and (6-4) photoproducts; and SI nuclease, which cuts the DNA where b a s e - p a i r i n g h a s been disrupted. In the past, the most widely used approach to measure strand breaks was alkaline sucrose-gradient centrifugation of prelabeled DNA (32). Recently, alkaline agarose gel elec trophoresis has been used (33). The size number and average molecular l e n g t h of t h e c l e a v e d DNA, a n d
hence the number of strand breaks, can be accurately determined by dig ital image analysis of the DNA in the gels following fluorescent staining by e t h i d i u m bromide (34). This tech nique permits detection of 3 cyclobu tadipyrimidine sites per 10 6 bases in as little as 3 0 - 5 0 ng of nonradioac tive DNA, which is e q u i v a l e n t to 0.3 fmol of cyclobutane-pyrimidine dimer. Using this methodology, Freeman and co-workers (35) determined an action spectrum for the frequency of cyclobutadipyrimidine formation in duced in the DNA of human skin per unit dose of UV radiation incident on the skin surface. The peak of the ac tion s p e c t r u m was observed to be n e a r 300 nm with a rapid falloff at both shorter and longer wavelengths. At 302 nm, 10 1 9 photons/cm 2 gener ated 1-10 e n z y m e - s e n s i t i v e sites (cyclobutadipyrimidines) per kilobase DNA in the skin of various indi viduals, whereas at 280 and 313 nm the yield of cyclobutane pyrimidine d i m e r s was approximately 10-fold lower. The decreased yield at wave lengths < 300 nm was attributed to light absorption by the upper layers of the skin. A related procedure has been de vised (36) to examine the extent of DNA photodamage in specific genes (Figure 5). DNA is recovered from
Enzyme-sensitive site assays These assays make use of the DNAnicking activity of r e p a i r enzymes
Figure 5. Experimental procedure for the detection of cyclobutadipyrimidines in specific genomic sequences.
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U V - i r r a d i a t e d cells a n d d i g e s t e d with restriction enzymes t h a t produce a gene fragment of known size. An aliquot of the DNA is then treated w i t h t h e UV e n d o n u c l e a s e . Both samples of DNA are subjected to alkaline gel electrophoresis and a n a lyzed by Southern blot hybridization with a specific probe for the gene restriction fragment of interest. The detected restriction fragments from DNA u n t r e a t e d w i t h UV e n d o n u clease (or from n o n i r r a d i a t e d control) are of a uniform length. On the other hand, the restriction fragments detected from the UV endonuclease t r e a t e d DNA h a v e a more or less random size (equal to or shorter than the full-length restriction fragment) and appear as a smear on the autoradiogram. This system has proven to be excellent for monitoring the cellular repair of damage in specific genes, because with increasing time of cellular repair before the DNA is extracted from the cells, one observes that the signal from the full-length fragment increases with a concomitant drop in the signal from the truncated fragments. Using this assay, Bohr and co-workers (36) were able to show t h a t t h e r e is preferential repair of DNA of t r a n s c r i p t i o n a l l y a c t i v e genes within the cells. Oxidative base damage sensitive to endonuclease III and FPG protein has been quantitated in photosensitized L1210 mouse leukemia cells by using a modified alkaline elution assay (37). Sequencing polyacrylamide gel technology provides a powerful tool by which to q u a n t i t a t e and locate base damage as either alkali labile modifications or DNA repair enzyme-sensitive sites within defined sequence oligonucleotides. An interesting application of this assay involves the determination of oxidative b a s e d a m a g e i n d u c e d in DNA by photoexcited 3 - carbethoxypsoralen and other furocoumarins (38). Future trends Two strategies are emerging for enhancing the detection levels of photoinduced and oxidative modifications in damaged DNA. The first involves the application of extremely sensitive i n s t r u m e n t a t i o n , for example, capillary gel electrophoresis coupled to laser fluorescence detection. The second entails the use of biochemical amplifying techniques such as t h e polymerase chain r e a c t i o n (PCR). PCR takes advantage of the doublestranded nature of DNA, in a process whereby specific sequences of denatured DNA are used as templates for
Figure 6. Detection of DNA damage by immuno-PCR. fresh DNA synthesis (39). The quant i t y of DNA of such a sequence should be effectively doubled in the course of each cycle of denaturation and synthesis, and hence only a few cycles are required to amplify a sequence 106-fold. Recently, in an elegant study, Pfeifer and co-workers (40) modified the PCR technique to examine the induction of (6-4) photoproducts in a specific sequence in cellular DNA. DNA was isolated from far-UV-irradiated m a m m a l i a n cells a n d c l e a v e d a t (6-4) photoproducts with piperidine (cyclobutadipyrimidines are not affected by this treatment). A specific fragment of the phosphoglycerate kin a s e gene was t h e n amplified and radiolabeled by a modified PCR procedure (19 cycles), and the lengths of the recovered fragments were determined by polyacrylamide gel electrophoresis. Among other things, they were able to show that the formation of (6-4) photoproducts was dependent on the nearest-neighbor bases, inhibited by the binding of a t r a n scription factor to the cellular DNA, and different for DNA derived from
active and inactive chromosomes. An e x c i t i n g n e w d e v e l o p m e n t , which should be applicable to any form of DNA damage for which antibodies can be prepared, is immunoPCR (41). The method makes use of a c h i m e r i c m o l e c u l e c o m p o s e d of s t r e p t a v i d i n coupled to p r o t e i n A (Figure 6). This molecule can bind to biotinylated products and immunoglobulin G through streptavidin and protein A moieties, respectively. Thus IgG antibodies bound to a n t i gen can be detected by addition of the chimera, followed by addition of a biotinylated DNA sequence. The signal is t h e n amplified by PCR of t h e DNA. T h i s t e c h n i q u e s h o u l d have a sensitivity far greater t h a n t h a t of existing a n t i g e n detection systems. References (1) Cadet, J.; Vigny, P. In Bioorganic Photochemistry; Morrison, H., Ed.; John Wiley and Sons: New York, 1990; Vol. 1, pp. 1-272. (2) Cadet, J. In DNA Adducts; Hemminki, K., Ed.; IARC Scientific Publication: Lyon, France, in press. (3) Cadet, J.; Anselmino, C ; Douki, T.;
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RŒPORT Voituriez, L. /. Photochem. Photobiol. B.: Biol. 1992, 15, 277-98. (4) Floyd, R. Α.; W a t s o n , J. J.; Wong, P. K.; Altmiller, D. H.; Rickard, R. C. Free Radical Res. Comm. 1986, 1, 163-71. (5) Wagner, J. R.; Hu, C - C ; Ames, Β. Ν. Proc. Natl Acad. Set. USA 1992,89, 3 3 8 0 84. (6) Berger, M.; C a d e t , J.; Berube, R.; L a n g l o i s R.; v a n Lier, J . E. / . Chromatogr. 1992, 593, 133-38. (7) Jajoo, H. K.; Burcham, P. C ; Goda, Y.; Blair, Ι. Α.; Marnett, L. J. Chem. Res. Toxicol. 1992, 5, 870-75. (8) Moysan, Α.; Vigny, P.; Dardalhon, M.; Averbeck, D.; Voituriez, L.; Cadet, J. / Photochem. Photobiol. 1988, 47, 803-08. (9) Wagner, J. R.; Berger, M.; Cadet, J.; van Lier, J. Ε. /. Chromatogr. 1990, 504, 191-96. (10) Fuciarelli, A. F.; Wegher, B. J.; Gaj e w s k i , E.; D i z d a r o g l u , M.; Blakely, W. F. Radiât. Res. 1989, 119, 2 1 9 - 3 1 . (11) Cadet, J.; Incardona, M-F.; Odin, F.; Molko, D.; Mouret, J - F . ; Polverelli, M.; F a u r e , H.; D u c r o s , V.; T r i p i e r , M.; Favier, A. In Postlabelling Methods for the Detection of UNA Damage; Phillips, D. H.; Castegnaro, M.; Bartsch, H., Eds.; IARC Scientific Publication: Lyon, France, in press. (12) Djuric, Z.; Luongo, D. Α.; H a r p e r , D. A. Chem. Res. Toxicol. 1991, 4, 6 8 7 - 9 1 . (13) Dizdaroglu, M. FEBS Lett. 1993, 315, 1-6. (14) Ramsey, R. S.; Ho, C-H. Anal. Biochem. 1989, 182, 4 2 4 - 3 1 . (15) A n n a n , R. S.; K r e s b a c h , G. M.; Giese, R. W.; Vouros, P. / . Chromatogr. 1989, 465, 285-96. (16) McCloskey, J. A ; Crain, P. F. Int. J. Mass Spectrom. Ion Processes 1992, 118/ 119, 593-615. (17) Randerath, K.; Reddy, M. V.; Gupta, R. C. Proc. Natl. Acad. Sci. USA 1981, 78, 6126-29. (18) Hegi, Μ. Ε.; Sagelsdorff, P.; Lutz, W. K. Carcinogenesis 1989, 10, 4 3 - 4 7 . (19) Weinfeld, M.; Soderlind, K-J.M. Bio chemistry 1991, 30, 1091-97. (20) Mouret, J - F . ; Odin, F.; Polverelli, M.; Cadet, J. Chem. Res. Toxicol. 1990, 3, 102-10. (21) W i n t e r s , Τ. Α.; Weinfeld, M.; J o r gensen, T. J. Nucleic Acids Res. 1992, 20, 2573-80. (22) Weinfeld, M.; Buchko, G. W. In Postlabelling Methods for the Detection ofDNA Damage; Phillips, D. H ; Castegnaro, M.; Bartsch, H , Eds.; IARC Scientific Pub lication: Lyon, France, in press. (23) Cadet, J.; Odin, F.; Mouret, J - F . ; Polverelli, M.; Audic, A ; Giacomoni, P.; Richard, M - J . ; Favier, A. Mutat. Res. 1992, 275, 343-54. (24) Frenkel, K ; Zhong, Z.; Wei, H.; Karkoszka, J.; Patel, U.; Rashid, K.; Georgescu, M.; Solomon, J. J. Anal. Biochem. 1991, 196, 126-36. (25) S h a r m a , M.; Box, H. C ; K e l m a n , D.J. Chem. Biol. Interact. 1990, 74, 107-17. (26) Levine, L.; Seeman, E. S.; Hammerschlag, E.; Van Vunakis, H. V. Science 1966, 153, 1666-67. (27) Mitchell, D. L.; Clarkson, J . M. / . Photochem. Photobiol. 1984, 40, 743-48. (28) Mori, T.; M a t s u n a g a , T.; C h a n g , C - C ; Trosko, J. E.; Nikaido, O. Mutat. Res. 1990, 236, 99-105. (29) Mizuno, T.; M a t s u n a g a , T.; I h a r a , M.; Nikaido, O. Mutat. Res. 1991, 254, 175-84. (30) Lesko, S. A ; Li, W.; Zheng, G.; Cal lahan, D.; Kaplan, D. S.; Midden, W. R.; Strickland, P. T. Carcinogenesis 1989, 10, 641-46.
(31) van der S c h a n s , G. P.; van Loon, A.A.W.; Groenendijk, R. H.; Baan, R. A. Int. J. Radial Biol. 1989, 55, 747-60. (32) P a t e r s o n , M. C. Adv. Radial Biol. 1978 7 1—53 (33) F r e e m a n , S. E.; B l a c k e t t , A. D.; M o n t e l e o n e , D. C ; S e t l o w , R. B . ; S u t h e r l a n d , B. M.; S u t h e r l a n d , J . C. Anal. Biochem. 1986, 158, 119-29. (34) S u t h e r l a n d , J. C ; Lin, B.; Monte leone, D. C ; Mugarevo, J.; Sutherland, B. M.; T r u n k , J. Anal. Biochem. 1987, 163, 446-57. (35) Freeman, S. E.; Hacham, H.; Gange, R. W.; Maytum, D. J.; Sutherland, J. C ; Sutherland, Β. Μ. Proc. Natl. Acad. Sci. USA 1989, 86, 5605-09. (36) Bohr, V. Α.; Okumoto, D. S.; H a nawalt, P. C. Proc. Natl. Acad. Sci. USA 1986, 83, 3830-33. (37) Epe, B.; Pflaum, M.; Boiteux, S. Mu tat. Res. 1993, 299, 135-145. (38) Sage, E.; Le Doan, T.; Boyer, V.; Helland, D. E.; Kittler, L.; Hélène, C ; Moustacchi, E . / . Mol. Biol. 1989, 209, 297-314. (39) Mullis, K.; Faloona, F.; Scharf, S.; S a i k i , R.; H o r n , G.; E r l i c h , H. Cold Spring Harbor Symp. Quant. Biol. 1986, 51 263—73 (40) Pfeifer, G. P.; Drouin, R.; Riggs, A. D.; Holmquist, G. P. Proc. Natl. Acad. Sci. USA 1991, 88, 1374-78. (41) Sano, T.; Smith, C. L.; Cantor, C. Science 1992, 258, 120-22.
Prepare yourself to meet the demands facing your lab today. Attend
' ALEXSM 93 The Analytical Laboratory Exposition and Conference
October 5-7,1993 Fashion Center San Francisco, CA USA • 200+ Exhibitors • Thousands of Products Jean Cadet obtained his Ph.D. in chemistry from the University of Grenoble in 1973. In 1968 he joined the French Atomic Energy Commission and heads the laboratory working on DNA damage. His research is focused on photochemical, radiation-induced, and oxidation reactions of DNA with emphasis on mechanistic aspects and DNA repair studies.
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Michael Weinfeld received his B.A. degree from Oxford University in 1978 and his Ph.D. from the University of London in 1982. His current research interests include oxidative DNA damage and its repair as well as enzyme recognition of DNA substrates.
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682 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
