(CANCER RESEARCH 48. 2156-2161. April 15, 1988] 06-Alkyldeoxyguanosine Detection by 32P-Postlabeling and Nucleotide Chromatographie Analysis Vincent L. Wilson,1 Ashis K. Basu,2 John M. Essigmann,2 Ruth A. Smith, and Curtis C. Harris Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [V. L. W., R. A. S., C. C. HJ; and Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02319 [A. K. B., J. M. EJ ABSTRACT The "P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quan- titation of O'-alkyl-2'-deoxyguanosine residues in DNA. Chromato graphie techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O'-alkyldeoxyguano- sine-3'-monophosphates by high-pressure liquid chromatography. Selec tive deoxyribonucleotide-3/-monophosphates (e.g., O'-alkyldeoxyguano- sine-3'-monophosphates) were then converted to labeled deoxyribonucle- otide-(5'-"P]monophosphates by 32P-postlabeling and nuclease PI treat ment and separated by two-dimensional thin layer chromatography. The 0*-methyl- and 0'-ethyl-2'-deoxyguanosine-3'-monophosphate nucleo- lides. and the respective 5'-monophosphates, were chemically synthe sized for standardization of these quantitative procedures. The quantita- tion of O'-methyl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O'-aIkyl-2'-deoxyguanosine residue per 10' and 1112'-deoxyguanosines. The limit of detection was less than one 0*-alkyl-2'-deoxyguanosine in III 2'-deoxyguanosine residues in a sample size of 100 Mgof DNA, i.e., approximately 10 pg of adduct. The guaniitaiiim of O'-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with ["C-MeyV-nitrosodimethylamine compared well with values obtained by both I4C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these "P-postlabeling and nucleotide Chromatographie procedures should be useful in monitoring human ex posure to methylating and ethylating carcinogens. INTRODUCTION The determination of chemical carcinogens as etiological factors in the occurrence of selected human cancers would be facilitated by the ability to assess not only the extent of exposure but also the biological damage (e.g., carcinogen-DNA adducts) subsequently produced in the tissues and cells of people exposed to carcinogens (1). Many techniques have been developed to detect minute levels of aromatic carcinogens in biological fluids and in cellular DNA. Immunoassay systems comprise the ma jority of these sensitive methods for the detection of polycyclic aromatic hydrocarbons (2-6), acetylaminofluorene (7), and af- latoxin BI (8, 9). The assessment of polycyclic aromatic hydro carbon-type carcinogens, other arylating carcinogens, and af- latoxin H, exposure has, however, been enhanced by the use of synchronous fluorescence spectrophotometric (10-12) and 32P- postlabeling methods (13, 14), which complement the radio- immunological techniques. The detection of alkyl-type DNA adducts such as O6-alkyldG3 Received 9/8/87; revised 12/28/87; accepted 1/19/88. 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. 1To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, NCI/NIH, Building 37, Room 2C20, Bethesda, MD 20892. * Supported by Grant CA-43066 from the NIH, Department of Health and Human Services. 'The abbreviations used are: O*-alkyldG, O'-alkyl-2'-deoxyguanosine; O6- mG, O*-methylguanine; O'-mdG, 0*-methyl-2'-deoxyguanosine; O'-etdG, O'- ethyl-2'-deoxyguanosine; 3'O*-alkyldGp, O'-alkyl-2'-deoxyguanosine-3'-mono- phosphate; 3'O'-mdGp, O'-methyl-2'-deoxyguanosine-3'-monophosphate; 3'O6- etdGp, O*-ethyl-2'-deoxyguanosine-3'-monophosphate; 3'dNp, 2'-deoxyribonu- cleotide-3'-monophosphate; S'pO'-alkyldG, O*-alkyl-2'-deoxyguanosine-5'- monophosphate;5'pO6-mdG, O6-methyl-2'-deoxyguanosine-5'-monophosphate; 5'pO*-etdG, O*-ethyl-2'-deoxyguanosine-5'-monophosphate; 5'pSmdC, 5- methyl-2'-deoxycytidine-5'-monophosphate; 5'pdN, 2'-deoxyribonucleotide-5'- monophosphate; III'I ( . high-pressure liquid chromatography; TLC, thin-layer chromatography. has been performed by high pressure liquid chromatography (HPLC) with fluorescence detection (15) and radioimmunolog- ical techniques (16-18). The determination of the levels of O6- alkyldG residues in human DNAs may be important due to the mutagenic potential of this adduct (19-21) and the strong correlation of DNA oxygen alkylation and the carcinogenic potency of agents (22-25). Human exposure to jY-nitroso com pounds and other alkylating agents occurs in tobacco smoke (24-26), industrial work environments and fossil fuels (27), and cancer chemotherapy (28). One problem that has arisen is that even monoclonal anti bodies generally recognize, at least to some extent, adducts other than the major adduct under study. Clearly, alternative and supporting methodologies that may corroborate the radio- immunoassay results are needed for the quantitation of alkyl DNA adducts. In response to these needs, we have modified the "P-postlabeling and nucleotide Chromatographie method ology developed by Randerath and coworkers (29) for the detection and analytical quantitation of O6-mdG and 06-etdG. MATERIALS AND METHODS Chemicals. The deoxyribonucleotides 5'pSmdC, 5'pdC, 5'pdT, 5'pdG, 5'pdA, 3'dCp, 3'dTp, 3'dGp, and 3'dAp were obtained from Pharmacia PL Biochemicals (Piscataway, NJ). JV-Nitrosodimethyla- mine was purchased from Sigma (St. Louis, MO) and [MC-MeJ/V- nitrosodimethylamine (14 mCi/mmol) was obtained from Amersham (Arlington Heights, IL). Radiolabeled (7-32P]ATP (>5000 Ci/mmol) was obtained from Amersham. 5'-Dimethoxytrityl-A'-isobutyryl-2'- deoxyguanosine was purchased from Cruachem, Inc. (Herndon, VA). Hydrazine hydrate, A'.W-dicyclohexylcarbodiimide (DCCI), 1,8-dia- zabicyclo[5.4.0]undec-7-ene (DBU), levulinic acid, triisopropylbenzene- sulfonyl chloride (TPS-C1), anhydrous trimethylamine (Mc,N), anhy drous pyridine, and 2-cyanoethyl phosphate (barium salt dihydrate) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Preparation of 3'- and S'-Monophosphate O'-Methyl- and O'-Ethyl- 2'-deoxyguanosine. The general synthetic route for 3'- and S'-mono- phosphates of 06-methyl- and 0'-ethyl-2'-deoxyguanosine is shown in Fig. 1. Completely protected derivatives of O'-alkyl-2'-deoxyguanosine (II) were prepared by a synthetic procedure developed by Jones and coworkers (30-32); facile sulfonylation of the 6 position of guanine by triisopropylbenzenesulfonyl chloride, followed by its displacement by anhydrous trimethylamine, generated an unstable 6-trimethylamino compound which, in turn, was converted to the 06-alkyl derivative (II) with a high yield. After purification, a portion of the nucleoside deriv ative (II) was detritylated by aqueous acid treatment to form III, whereas the rest of the derivative II was treated with hydrazine to hydrolyze the 3'-levulinate group yielding IV. Phosphorylation of the 3'-hydroxyl functionality was performed under anhydrous conditions by pyri- diniimi /¡-cyanoctliylphosphate in the presence of A^yv'-dicyclohexyl- carbodiimide in pyridine following the method of Tener (33). Depro- tection of III was carried out by the alkoxide ion in the respective alcohol in l,8-diazabicyclo[S.4.0]undec-7-ene for 2 days (31); deblock ing of IV, in addition, required detritylation. The 0*-alkyl-2'-deoxy- guanosine phosphates, thus produced, were purified on a Whatman Partisi! SAX column (linear gradient of 1-300 m\i potassium phos phate buffer (pH 5.3) over 60 min, at a flow rate of 1.5 ml/min) followed by desalting on a Ci8 Sep-Pak (Waters) cartridge. The Sep- Pak cartridge was prepared by washing sequentially with 50% acetoni- 2156 on March 14, 2016. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from