Chemically Induced Depurination Under Physiological Conditions'

(CANCER RESEARCH 58. 222-225, January 15. 1998]

Advances in Brief

Highly Sensitive Apurinic/Apyrimidinic Site Assay Can Detect Spontaneous and Chemically Induced Depurination under Physiological Conditions'

Jun Nakamura, Vernon E. Walker,2 Patricia B. Upton, Su-Yin Chiang, Yoke W. Kow,3 and James A. Swenberg4

Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, North Carolina 27599 [J. N., V. E. W., P. B. U., S-I'. C., J. A. S.], and Department ofMicrobiology and Molecular Genetics, Markey Centerfor Molecular Genetics, University of Vermont, Burlington, Vermont 05405 (1'. W. K.J

Abstract tive to measure low numbers of AP sites, or difficult to conduct. Re cently, a novel reagent for measuring AP sites was prepared by reacting One of the most prevalent lesions in DNA is the apurinic/apyrimidinic O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence (AP) site, which is derived from the cleavage of the N-glycosyl bond by of carboiimide (15). This reagent, called ARP, is a specific biotin-tagged DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonudeases during the process of base excision repair; however, an probe that reacts with aldehydic ring-opened AP sites (15, 16). The imbalance in this DNA repair system may cause mutations as well as cell number ofbiotin-tagged AP sites can then be determined colorimetrically death. We have established a sensitive and convenient slot-blot method to by an ELISA-like assay. The present study reports a newly developed and detect AP sites in genomic DNA using a novel aldehyde reactive probe highly sensitive method for the detection of AP sites in genomic DNA by (ARP), which reacts with the aldehydic group ofring-opened AP sites. The a combination of ARP and slot-blot techniques. reaction of 1 msi of ARP with 15 @sgofgenomic DNA containing AP sites at 37Â°Cwas completed within 1 mm. The AP site-ARP complex was Materials and Methods remarkably stable during incubation in TE buffer, even at 100Â°Cfor60 asia. The sensitivity of this assay enables detection of 2.4 AP sites per tO7 Aldehyde Reactive Probe-Slot-BloL The protocolof ASB uses the slot bases. By using this ARP-slot-blot assay, the rate of spontaneous depuri blot method and ARP to measure AP sites (15). Fifteen p.g of DNA in 150 @l nation of calf thymus DNA was determined. Under physiological condi of PBS (137 mM NaC1, 2.7 nmi KC1,4.3 mxi NaH2PO47H2O, and 1.4 mxi tions, AP sites were increased at 1.54 AP sites/tOe nucieotides/day (9000 K@'@2PÂ°[email protected]) was incubated with 1 mM ARP (Dojindo Laboratories, AP sites/cell/day). This highly sensitive assay allows us to determine the Kumamoto, Japan) at 37Â°Cfor10 mm. The number of AP sites in the internal endogenous level of AP sites in genomic DNA, as well as to investigate standard DNA was determined by the microtiter plate method with ARP by Dr. whether DNA-damaging agents cause imbalances of base excision/AP Kubo (University of Osaka Prefecture, SaICai,Japan).After precipitation using endonuclease repair in vivo and in vitro. cold ethanol, DNA was washed with 70% ethanol and resuspended in TE buffer (10 mMTns-HC1, I misiEDTA, pH 7.2) at 3 @g/l00p1 The internal Introduction standard was serially diluted with calf thymus DNA (3 @g/l00 @lTEbuffer). DNA was heat-denatured at 100Â°Cfor 5 mm, quickly chilled on ice, and mixed Various DNA adducts are produced by electophilic chemicals such with an equal amount of 2 Mammonium acetate. The single-stranded DNA was as alkylating agents. In addition, highly sensitive methods have en then immobilized on a BAS-85 NC membrane (Schleicher and Schuell) using a Minifold II vacuum filter device (Schleicher and Schuell). The slots were abled detection of endogenous adducts in DNA extracted from tissues rinsed with 200 p1 of 1 M ammonium acetate. The NC membrane was soaked of experimental animals as well as humans (1). Many of these DNA with 5X SSC (0.75 M NaC1, 0.075 M tnsodium citrate) at 37Â°Cfor 15 rain and lesions are repaired by a base excision repair pathway (2â€”6).During then dried and baked in a vacuum oven at 80Â°Cfor1â€”2h.The membrane was the first process of base excision repair, DNA glycosylase cleaves a preincubated with 10 ml ofTris-NaC1 buffer containing BSA [20 mM Tris-HCI modified DNA base at the glycosyl bond, resulting in formation of an (pH 7.5), 0.1 MNaC1,I mxiEDTA, 0.5% casein, 0.25% BSA, and 0.1%Tween AP5 site. AP sites can also be caused by spontaneous depurination of 20] at room temperature for 1 h. The NC filter was then incubated in the same labile DNA adducts as well as unmodified bases. solution containing streptavidin-conjugated horseradish peroxidase (BioGenix) at room temperature for 30â€”45mm. After rinsing the NC membrane with Formation of AP sites is a relatively frequent event in chromosomal washing buffer (0.26 MNaC1, 1 mxi EDTA, 20 mM Tris-HC1, and 0.1% Tween DNA under physiological conditions (7). AP sites inhibit DNA rep 20, pH 7.5) for 15 mm, the enzymatic activity on the membrane was visualized lication and also result in base substitution mutations and loss of by the ECL reagents (Amersham Corp.). The NC filter was then exposed to genetic integrity (8). In a recent study, it was proposed that an X-ray film (Kodak XAR 5X; Kodak) for 5â€”15s.The developed film was imbalance in the base excision repair pathway causes mutational analyzed using a Ultrascan XL scanning densitometor (Pharmacia) and events and may play an important role in carcinogenesis (9). GeIScan XL software (Pharmacia). Quantitation was based on comparisons to internal standard DNA containing the known amount of AP sites. Several methods are presently available to detect AP sites (10â€”16); AP Site Preparation by Heat/Acid Condition. AP sites wereproducedin however, these methods are either not direct measurements, insensi a calf thymus DNA by heat/acid-buffer solution. Intact calf thymus DNA (Sigma Chemical Co.) or calf thymus DNA pretreated with 5 mr@tmethoxy Received 9/15/97; accepted 12/1/97. amine (Sigma) was added to sodium citrate buffer (10 mt@isodium citrate The costs of publication of this article were defrayed in part by the payment of page containing 10 mM NaH2PO4 and 10 mM NaCl, pH 5.0) and held at 70Â°Cfor charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. various lengths of time. The reaction was stopped by chilling rapidly on ice, I Funded in part by Grant P42-ES05948 from NIEHS Superfund Basic Research and the DNA was then precipitated with cold ethanol, washed once with 70% Program and a grant from the Chemical Manufacturers Association. ethanol, dried, and resuspended in sterilized distilled water. 2 Present address: New York State Department of Health, Wadsworth Center for Treatment with hAPE and NaOH. Heat/acid-treated calf thymus DNA Laboratories and Research, Empire State Plaza, P. 0. Box 509, Albany, NY 12201-0509. (15 @Lg)and 23 ng of hAPE (a gift from Dr. M. Kelley, Indiana University, 3 Present address: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Ave., Atlanta, GA 30335. Indianapolis, Indiana) in 67.5 @lof10 m@iTris-HC1IKOHbuffer (pH 7.5) 4 To whom requests for reprints should be addressed, at Department of Environmental containing 50 mMNaCl, 5 mMMgC12were incubated at 37Â°Cfor30 mm. For Sciences and Engineering, The University of North Carolina, CB# 7400, Chapel Hill, NC alkaline treatment, 1/10 volume of 2 MNaOH was added to the above reaction 27599-7400. mixture and then incubated at 37Â°Cfor 15 mm. The samples were precipitated 5 The abbreviations used are: AP, apurinic/apyrimidinic; ARP, aldehyde reactive probe; ASB, aldehyde reactive probe-slot-blot; NC, nitrocellulose; hAPE, human AP with cold ethanol as described above, followed by resuspension with PBS for endonuclease; MMS, methyl methanesulfonate. ASB assay. 222

AP Site Induction by Treatment with MMS. H2E1 cells, a human B-lymphoblastoid line immortalized with EBV expressing human Cyp 2El, A were generously provided by Gentest Corp. H2E1 cells were grown in RPM! 1640 supplemented with L-glutamine, HEPES buffer, NaI-1C03,1-histidinol, and 10%FCS (17). Cells (5.0 X l0@)perml were exposed in triplicate to MMS (0.05â€”1.5 mM) at 37Â°C for 24 h. Following treatment, the cells were pelleted and washed once by spinning for 4 mm at 1000 rpm. For determining toxicity of treatment, the cells were stained by trypan blue. The cell pellets were then frozen at â€”70Â°Cbeforeusing. DNA Extraction. DNA was extractedby a proceduremodified from the method reported by Gupta (18). After thawing the frozen cells, the pellets were incubated in lysis buffer (Applied Biosystem) overnight at 4Â°Cwithproteinase K (500 @g/ml,AppliedBiosystem). DNA was then extractedtwice with a mixture ofphenol/chloroform/water(Applied Biosystem) at 4Â°Cand once with Sevag (chloroform:isoamyl alcohol, 24:1), followed by ethanol precipitation. The extracted DNA was resuspended in sterilized PBS (pH 7.4) and incubated at 37Â°Cfor30 rein with a mixture of RNase Tl (50 units/mI) and RNase A (100 @.tg/ml).After extraction with Sevag, DNA was precipitated with ethanol and then resuspended in sterilized distilled water. The DNA solution was stocked at â€”70Â°CforASB assay.

Results

Sensitivity of the ASB Method. Heat/acid-treated calf thymus DNA containing 800 AP sites/106 nucleotides, incubated with 1 nmi ARP, was serially diluted with unmodified calf thymm DNA. A typical X-ray film from the ASB assay with 3 @tgofDNA/slot is shown in Fig. 1A. The slot density decreased with dilution of ARP-complex. The relationship between density, as measured by a scanning densitometer, and the concentration of AP sites is shown Fig. lB. The sensitivity of B ASB assay was typically 0.24 AP sitesll06 nucleotides. 10 Optimum Conditions ofASB. The ability of ARP reagent to react with AP sites in calf thymus DNA was examined. The reactivity was 8 measured by incubation with various concentrations of ARP ranging from 1 @.LMto10 mM, as well as different reaction times. The ARP 0 Cl) reaction at 37Â°Cfor 10 mm was enhanced by increasing the concentration 6 ofARP reagent, which reached saturationat 1 mi@iormore (Fig. 2A). The rate of reaction of 1 mr@iARP to AP sites was extremely rapid, with ARP incorporation into DNA accomplished within 1 mm at 37Â°C. U) C 4 Before immobilizing DNA on the NC membrane, it was necessary U) to denature the DNA after ARP conjugation. To evaluate the influence of denaturation on the stability of the ARP complex, the persistence of 2 the ARP complex at various incubation times at 100Â°Cwas measured using DNA samples containing 60, 120, and 800 AP sites/106 nude otides. The ARP complex was stable at 100Â°Cforat least 60 mm. We 1000500 100 50 10 5 1 0.5 varied the amount of DNA loaded on the NC membrane to study the AP sites! 106Nucleotides response of the ASB assay. A DNA sample containing 800 AP sites/106 nucleotides was serially diluted with TE buffer, and different Fig. 1. A, typical X-ray film in ASB assay. Three @agofheat/acid-treated calf thymus DNA per slot (standard DNA samples) was loaded on NC membrane. The slots with 3 ,.@g amounts of DNA were applied to the NC filter. We observed a linear of DNA containing, from top to bottom, 800, 240, 80, 24, 8, 2.4, 0.8, 0.24, 0.08, and 0 AP relationship between slot density and the amount of DNA (Fig. 2B). sites per 106 nucleotides, respectively. (DNA containing 0 AP sites/106 nucleotides was Effect ofhAPE on ASB Assay. To examine whether AP sites were ARP-unexposed calf thymus DNA.) B, scanning densitometric data of internal standard curve of ASB shown in Fig. (A. The mean peak areas derived from duplicate slots were detectable with the ASB assay after incision by AP endonuclease, calf plotted against concentration of AP sites in a double-logarithmic scale. thymus DNA treated with heat/acid-solution was incubated with hAPE, which incises the DNA 5' to the AP site. After 30 mm of incubation, hAPE reduced AP sites in the DNA approximately 28% (Fig. 3). In amine-pretreated calf thymus DNA was incubated at 37Â°Cin PBS (pH addition, the combination of hAPE and 0.2 M NaOH, which cleaves the 7.4) for up to 10 days. The increase in AP sites was proportional to the DNA 5' and 3' to the AP site, decreased the number of AP sites 70%. In incubation time. These data showed that approximately 1.54 AP sites/106 contrast, no reduction of AP sites was observed after treatment with nucleotides were introduced per day (Fig. 4B). 0.2 MNaOH, which incises DNA 3' to the AP site. AP Site Induction by MMS. H2E1 cells were exposed to MMS at AP Site Induction at Heat/Acid and Physiological Conditions. different concentrations ranging from 0.05 to 1.5 mi@iat37Â°Cfor 24 h. AP sites in calf thymus DNA were reduced by treatment with 5 mr@i Four AP sites/l06 nucleotides were detected in control H2E1 cells. AP methoxyamine and then created by incubating in sodium citrate buffer sites in DNA increased two and five times compared with control at (pH 5.0) at 70Â°Cfor up to 150 s. The number of AP sites increased in 0.5 and 1.5 mM, respectively (Fig. 5). In contrast, there was no proportion to the length of incubation. Linear regression revealed that increase in AP sites at 0. 15 m@ior less. In this experiment, cytotox approximately 10 AP sites/l06 nucleotides were produced per rain by icity evaluated by trypan blue dye exclusion showed high survival incubation at 70Â°Cat pH 5.0 (Fig. 4A). To determine the rate of spon rates (95% or more) after exposure to 0.05â€”0.5 mi@iMMS. Cell taneous depurmnation of DNA under physiological conditions, methoxy survival was more than 90% following exposure to 1.5 mtvi. 223

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. ARP-SLOT-BLOTASSAYTO DETECTAP SITES A cause there was no reaction between normal nucleosides and ARP during 5 a 4-day incubation period at 37Â°C(16), the number of AP sites detected in the control H2E1 cells appears to be due to endogenous AP sites in genomic DNA. Therefore, these data indicate that the sensitivity of the

4.' 0 0.5 I.Cl) 0

0 U) 0.1 0 C U) 0.05

0.01 @1 0.005 0.001 0.01 0.1 1 10 Concentration of ARP Reagent (mM)

B 7 0 No Treatment hAPE hAPE+NaOH NaOH Fig. 3. The effect of hAPE and NaOH on ASB assay. DNA incision reactions were 6 described in â€œMaterialsand Methods.â€• After the cleavage reaction with hAPE and/or NaOH followed by ethanol precipitation, AP sites in the DNA were measured by ASB assay. The data represent the mean of three to six independent determinants. Bars, SD. 0 5 (I) 0 4 A Cl) C U) U) 3 U)

4.' 0 2 U) C.) I z 3 I 0.3 0.1 0.03 DNA on Slot (ug) U)

Fig. 2. A, conjugation of ARP into heat/acid-treated calf thymus DNA containing three Cl) AP sites per l0@nucleotides as a function of the ARP concentration (0.1 @.tMto10 nwt). Constant 3 @agofDNA was incubated with various concentrations of ARP at 37Â°Cfor 10 mm. The mean peak areas derived from duplicate slots were plotted against a concentra tion of AP sites in a single-logarithmic scale. B, different amounts of ARP-conjugated calf thymus DNA containing 800 AP sites per 106 nucleotides (0, 03, 0.1, 0.3, 1, and 3 @gof 0 50 100 150 200 DNA/slot) was loaded on the NC membrane. Incubation Time (seconds) B Discussion 35 U) Sensitivity of ASB Method. The present study reports the devel U) opment of a highly sensitive, specific, and convenient method to 4.' 0 detect AP sites in genomic DNA at a detection limit of 0.24 AP U) 25 sites/l06 nucleotides. The sensitivity of the ASB assay, which is based C) on the combination of a slot-blot method and ARP reagent, is one or C two orders of magnitude higher than presently available methods of detecting AP sites (10â€”16).It has been estimated that the rate of AP site formation is 10,000 per cell per day in mammalian cells (7). In );15 this previous report, the rate of depurination of double-stranded DNA was estimated using the depurination rate of DNA at 70Â°Cand physical chemistry. This methodology was not sensitive enough to detect directly the slow rate of depurination at 37Â°Cand pH 7.4. In the 0 2 4 6 8 10 12 present study, the ASB assay clearly demonstrated spontaneous in Incubation Time (days) creases in AP sites, which are due to depurmnation, at 1.54 AP sites/l06 nucleotides/day (9,000 AP sites/cell/day) at 37Â°Cand pH 7.4. The num Fig. 4. A, AP site induction in calf thymus DNA by heat/acid treatment. Calf thymus DNA AP sites were reduced by treatment with 5 msi methoxyamine. After DNA her of AP sites present in genomic DNA under physiological conditions precipitation, the DNA was then incubated at 70Â°CatpH 5.0 for up to 156 a. AP sites in or AP site kinetics following exposure to low levels of DNA-damaging DNA were measured using ASB. Each point represents the mean of three independent agents is not known, due in part to the instability of AP sites and determinants; bars, SD. B, AP site formation in calf thymus DNA under physiological conditions. Methoxyamine-pretreated calf thymus DNA was incubated at 37CCand pH 7.4 insensitivity of AP site assays. In this report, we demonstrated four AP for up to 10 days. AP sites in DNA were measured using ASB. Each point represents the sites/106 nucleotides in control H2E1 cells (23,000 AP sites/cell). Be mean of three independent determinants; bars, SD. 224

30 compounds in vivo is not fully understood. This highly sensitive and simple method to detect AP sites should allow us to investigate AP site 25 kinetics and the balance between glycosylase and AP endonuclease 0 0 activities. In addition, this ASB method may lead to a better understand ing of the significance of the base excision repair pathway in carcino I20 genesis as well as the biological importance of endogenously and exog enously derived AP sites. 0 15 0 Acknowledgments j 10 We thank Dr. Kihei Kubo for determination of AP sites in our internal standard DNA and technical assistance. We also thank Dr. M. Kelley, who 5 supplied the hAPE. The critical reading of the manuscript by Dr. David La is acknowledged. 0 0.05 0.15 0.5 References Concentration MMS (mM) I. La, D. K., and Swenberg, J. A. DNA adducts: biological markers of exposure and Fig. 5. AP site induction in H2EI cells by MMS. H2E1 cells were exposed to MMS potential applications to risk assessment. Mutat. Res., 365: 129â€”146,1996. (0.05â€”1.5mM) at 37Â°Cfor 24 h. Each point represents the mean of three independent 2. Bessho, 1., Roy, R., Yamamoto, K., Kasai, H., Nishimura, S., Tano, K., and Mitra, determinants; bars, SD. S. Repair of 8-hydroxyguanine in DNA by mammalian N-methylpurine-DNA glyco sylase. Proc. Nail. Acad. Sci. USA, 90: 8901â€”8904,1993. 3. O'Connor, 1. R. Purification and characterization of human 3-methyladenine-DNA glycosylase. Nucleic Acids Ret., 21: 5561â€”5569,1993. ASB assay is sufficient to measure endogenous and chemically induced 4. Rydberg. B., Qiu, Z. H., Dosanjh, M. K., and Singer, B. Partial purification of a AP sites in genomic DNA. We are presently measuring the amount of human DNA glycosylase acting on the cyclic carcinogen adduct l,M@-ethenodeoxya endogenous AP sites using the ASB method in genomic DNA from denosine. Cancer Res., 52: 1377â€”1379,1992. 5. Dosanjh, M. K., Chenna, A., Kin, E., Fraenkel-Conrat, H., Samson, L., and Singer, B. different organs of different species. All four known cyclic adducts formed in DNA by the vinyl chloride metabolite To examine whether this assay can detect AP sites after cleavage of chloroacetaldehyde are released by a human DNA glycosylase. Proc. Natl. Acad. Sd. DNA 5' or 3' to an AP site, heat/acid-treated DNA was incubated with USA, 9/: 1024-1028, 1994. 6. Dosanjh, M. K., Roy, R., Mitra, S., and Singer, B. I,M'-ethenoadenine is preferred hAPE and/or NaOH at 37Â°C.There was no reduction in the number over 3-methyladenine as a substrate by a cloned human N-methylpurine-DNA gly of AP sites in the DNA following 3' cleavage to the AP site by (3-dim cosylase (3-methyladenine-DNA glycosylase). Biochemistry, 33: 1624â€”1628, 1994. ination with NaOH (19). In contrast, 5' incision by hAPE and both 5' and 7. Lindahl, T., and Nyberg, B. Rate of depurination of native deoxyribonucleic acid. Biochemistry, II: 3610â€”3618,1972. 3' nicks to AP sites by combination of hAPE and NaOH reduced the 8. Loeb, L. A., and Preston, B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu. number of AP sites by 28 and 70%, respectively. The marked decrease in Rev. Genet., 20: 201-230, 1986. the number of AP sites appears to be due to the release of AP sites from 9. Coquerelle, T., Dosch, J., and Kaina, B. Overexpression of N-methylpurine-DNA the DNA back bone. The reason why hAPE treatment alone reduced the glycosylase in Chinese hamster ovary cells renders them more sensitive to the production of chromosomal aberrations by methylating agentsâ€”acase of imbalanced number ofAP sites on the DNA may be due to the spontaneous induction DNA repair. Mutat. Res., 336: 9-17, 1995. of nicks 3' to AP sites during the incubation of DNA with heat/acid 10. Brent, T. P., Teebor, G. w., and Duker, N. J. Lesions in alkylated DNA determined solution. This experiment suggests that this assay may also be applicable by susceptibility to alkali, apurinid endonuclease or N-glycosyla.se. In: P. C. Hanawalt, E. C. Friedberg, and C. F. Fox (edt.), DNA Repair Mechanisms, pp. to assessing AP endonuclease activity as well as determining the location 19â€”22.New York: Academic Press, 1978. of cleavage relative to AP sites. I 1. Kohn, K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurement of Advantages of ASB Assay. As described above, the ASB assay is strand breaks and crosslinks in DNA by alkaline elution. In: E. C. Friedberg and P. C. Hanawalt (edt.), DNA Repair: A Laboratory Manual of Research Procedures, Vol. 1, a highly sensitive and specific method to detect AP sites. There are Part B, pp. 379â€”401.NewYork: Marcel Dekker, 1981. other advantages of the ASB over other methods for quantitation of 12. Weinfeld, M., Liuzzi, M., and Paterson, M. C. Response of phage T4 polynucleotide AP sites. This method uses small amounts of genomic DNA (15 @.tg) kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry, 29: 1737â€”1743,1990. isolated from any type of cells and tissues or enzyme-digested DNA 13. Talpaert-Borle, M., and Liuzzi, M. Reaction of apurinic/apyrimidinic sites with fragments with length more than 300 nucleotides without any prela [4C]methoxyamine. A method for the quantitative assay of AP sites in DNA. beling process. No radioactive materials are required, and 28 samples Biochim. Biophys. Acta, 740: 410â€”416, 1983. 14. Chen, B-X., Kubo, K., Ide, H., Erlanger, B. F., Wallace, S. S., and Kow, Y. W. can be analyzed in 1 or 2 days. Moreover, all materials required for Properties of a monoclonal antibody for the detection of abasic sites, a common DNA the ASB assay including ARP reagent are presently commercially lesion. Mutat. Res., 273: 253â€”261,1992. 15. Kubo, K., Ide, H., Wallace, S. S., and Kow, Y. W. A nobel, sensitive, and specific available. The ASB assay will be useful for measuring the amount of assay for abasic sites, the most commonly produced DNA lesion. Biochemistry, 31: heat-labile DNA adducts as well as DNA glycosylase activity. 3703â€”3708,1992. Biological Significance of AP Sites. The present study demon 16. Ide, H., Akamatsu, K., Kimura, Y., Michiue, K., Makino, K., Asaeda, A., Takamori, Y., and Kubo, K. Synthesis and damage specificity of a novel probe for the detection strated that the ASB assay can detect even a slight increase in AP sites of abasic sites in DNA. Biochemistry, 32: 8276â€”8283,1993. caused by heat/acid treatment or MMS. The formation of AP sites can 17. Chiang, S-Y., Swenberg, J. A., Weisman, W. H., and Skopek, T. R. Mutagenicity of lead to the induction of base substitution mutations or inhibition of DNA vinyl chloride and its reactive metabolites, chloroethylene oxide and chloroacetalde hyde, in a metabolically competent B-lymphoblastoid line. Carcinogenesis (Lond.), replication (8). Recently, it has been proposed that an imbalance in the 18: 31â€”36,1997. base excision repair pathway may play an important role in carcinogen 18. Gupta. R. C. Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminoflu esis (9). For example, overexpressionof N-methylpurine-DNAglycosy oreneto repetitivesequencesofrat liverDNAin vivo.Proc.NatI.Acad.Sci.USA, 81: 6943â€”6947, 1984. lase causes enhanced chromosomal aberrations induced by methylating 19. Bailly, V., Verly, W. G., O'Connor, T., and Laval, J. Mechanism of DNA strand agents in Chinese hamster ovary cells (9); yeast strains deficient in apnl, nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidinel DNA glycosylase. Biochem. J., 262: 581-589, 1989. the major AP endonuclease of Saccharomyces cerevisiae, have a sub 20. Ramotar, D., Popoff, S. C., Gralla, E. B., and Demple, B. Cellular role of yeast Apn I stantially elevated frequency of spontaneous mutations (20). A combina apurinic endonucleasel3'-diesterase: repair of oxidative and alkylation DNA damage tion ofN-methylpurine-DNA glycosylase overexpression or AP endonu and control of spontaneous mutation. Mol. Cell. Biol., I 1: 4537â€”4544, 1991. 21. Xiao, W., and Samson, L. In vivo evidence for endogenous DNA alkylation damage clease deficiency markedly increases in the spontaneous mutation rate in as a source of spontaneous mutation in eukaryotic cells. Proc. Nail. Acad. Sci. USA, yeasts (21). However, the kinetics ofAP sites after exposure to mutagenic 90:2117-2121,1993. 225

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. Highly Sensitive Apurinic/Apyrimidinic Site Assay Can Detect Spontaneous and Chemically Induced Depurination under Physiological Conditions

Jun Nakamura, Vernon E. Walker, Patricia B. Upton, et al.

Cancer Res 1998;58:222-225.

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