(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 One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonudeases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group ofring-opened AP sites. The reaction of 1 msi of ARP with 15 @sg of genomic DNA containing AP sites at 37°Cwas completed within 1 mm. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100°C for 60 asia. The sensitivity of this assay enables detection of 2.4 AP sites per tO7 bases. By using this ARP-slot-blot assay, the rate of spontaneous depuri nation of calf thymus DNA was determined. Under physiological condi tions, AP sites were increased at 1.54 AP sites/tOe nucieotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro. Introduction Various DNA adducts are produced by electophilic chemicals such as alkylating agents. In addition, highly sensitive methods have en abled detection of endogenous adducts in DNA extracted from tissues of experimental animals as well as humans (1). Many of these DNA lesions are repaired by a base excision repair pathway (2—6).During the first process of base excision repair, DNA glycosylase cleaves a modified DNA base at the glycosyl bond, resulting in formation of an AP5 site. AP sites can also be caused by spontaneous depurination of labile DNA adducts as well as unmodified bases. Formation of AP sites is a relatively frequent event in chromosomal DNA under physiological conditions (7). AP sites inhibit DNA rep lication and also result in base substitution mutations and loss of genetic integrity (8). In a recent study, it was proposed that an imbalance in the base excision repair pathway causes mutational events and may play an important role in carcinogenesis (9). Several methods are presently available to detect AP sites (10—16); however, these methods are either not direct measurements, insensi Received 9/15/97; accepted 12/1/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Funded in part by Grant P42-ES05948 from NIEHS Superfund Basic Research Program and a grant from the Chemical Manufacturers Association. 2 Present address: New York State Department of Health, Wadsworth Center for Laboratories and Research, Empire State Plaza, P. 0. Box 509, Albany, NY 12201-0509. 3 Present address: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Ave., Atlanta, GA 30335. 4 To whom requests for reprints should be addressed, at Department of Environmental Sciences and Engineering, The University of North Carolina, CB# 7400, Chapel Hill, NC 27599-7400. 5 The abbreviations used are: AP, apurinic/apyrimidinic; ARP, aldehyde reactive probe; ASB, aldehyde reactive probe-slot-blot; NC, nitrocellulose; hAPE, human AP endonuclease; MMS, methyl methanesulfonate. 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 O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carboiimide (15). This reagent, called ARP, is a specific biotin-tagged probe that reacts with aldehydic ring-opened AP sites (15, 16). The number ofbiotin-tagged AP sites can then be determined colorimetrically by an ELISA-like assay. The present study reports a newly developed and highly sensitive method for the detection of AP sites in genomic DNA by a combination of ARP and slot-blot techniques. Materials and Methods Aldehyde Reactive Probe-Slot-BloL The protocol of ASB uses the slot blot method and ARP to measure AP sites (15). Fifteen p.g of DNA in 150 @l of PBS (137 mM NaC1, 2.7 nmi KC1, 4.3 mxi NaH2PO47H2O, and 1.4 mxi K@'@2P°4@ pH 7.4) was incubated with 1 mM ARP (Dojindo Laboratories, Kumamoto, Japan) at 37°C for 10 mm. The number of AP sites in the internal standard DNA was determined by the microtiter plate method with ARP by Dr. Kubo (University of Osaka Prefecture, SaICai, Japan). After precipitation using 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 standard was serially diluted with calf thymus DNA (3 @g/l00 @l TE buffer). DNA was heat-denatured at 100°Cfor 5 mm, quickly chilled on ice, and mixed with an equal amount of 2 M ammonium acetate. The single-stranded DNA was then immobilized on a BAS-85 NC membrane (Schleicher and Schuell) using a Minifold II vacuum filter device (Schleicher and Schuell). The slots were rinsed with 200 p1 of 1 M ammonium acetate. The NC membrane was soaked with 5X SSC (0.75 M NaC1, 0.075 M tnsodium citrate) at 37°C for 15 rain and then dried and baked in a vacuum oven at 80°C for 1—2 h. The membrane was preincubated with 10 ml ofTris-NaC1 buffer containing BSA [20 mM Tris-HCI (pH 7.5), 0.1 MNaC1, I mxi EDTA, 0.5% casein, 0.25% BSA, and 0. 1% Tween 20] at room temperature for 1 h. The NC filter was then incubated in the same solution containing streptavidin-conjugated horseradish peroxidase (BioGenix) at room temperature for 30—45mm. After rinsing the NC membrane with washing buffer (0.26 M NaC1, 1 mxi EDTA, 20 mM Tris-HC1, and 0.1% Tween 20, pH 7.5) for 15 mm, the enzymatic activity on the membrane was visualized by the ECL reagents (Amersham Corp.). The NC filter was then exposed to X-ray film (Kodak XAR 5X; Kodak) for 5—15 s. The developed film was analyzed using a Ultrascan XL scanning densitometor (Pharmacia) and GeIScan XL software (Pharmacia). Quantitation was based on comparisons to internal standard DNA containing the known amount of AP sites. AP Site Preparation by Heat/Acid Condition. AP sites wereproducedin 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 amine (Sigma) was added to sodium citrate buffer (10 mt@isodium citrate containing 10 mM NaH2PO4 and 10 mM NaCl, pH 5.0) and held at 70°Cfor various lengths of time. The reaction was stopped by chilling rapidly on ice, and the DNA was then precipitated with cold ethanol, washed once with 70% ethanol, dried, and resuspended in sterilized distilled water. Treatment with hAPE and NaOH. Heat/acid-treated calf thymus DNA (15 @Lg)and 23 ng of hAPE (a gift from Dr. M. Kelley, Indiana University, Indianapolis, Indiana) in 67.5 @l of 10 m@iTris-HC1IKOHbuffer (pH 7.5) containing 50 mMNaCl, 5 mMMgC12were incubated at 37°C for 30 mm. For alkaline treatment, 1/10 volume of 2 MNaOH was added to the above reaction mixture and then incubated at 37°Cfor 15 mm. The samples were precipitated with cold ethanol as described above, followed by resuspension with PBS for ASB assay. 222 Research. on August 12, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from