Proc. Natd Acad. Sci. USA Vol. 78, No. 10, pp. 6126-6129, October 1981 Biochemistry 32P-Labeling test for DNA damage (environmental carcinoens/mutaxens/polvethyleneimine-celulose/din-laver chromatonranhv) KURT RANDERATH, M. VIJAYARAJ REDDY, AND RAMESH C. GUPTA Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030 Communicated by Paul C. Zamecnik, July 6, 1981 ABSTRACT Covalent adducts formed by the reaction ofDNA MATERIALS AND METHODS withchemicalcarcinogens and mutagens maybedetected bya32P- labeling test. DNA preparations exposed to chemicals known to Materials. N-Methyl-N-nitrosourea, dimethyl sulfate, pro- bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sul- pylene oxide (Aldrich), formaldehyde (Mallinckrodt), mechlor- fate, formaldehyde, -propiolactone, propylene oxide, strepto- ethamine (a nitrogen mustard; Merck Sharp & Dohme), strep- zotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosou- tozotocin (Upjohn), 1,3-bis(2-chloroethyl)-1-nitrosourea (Bristol rea] were digested to a mixture ofdeoxynucleoside 3'-monophos- Laboratories), and (3-propiolactone (Sigma) were used without phates by incubation with micrococcal endonuclease (EC 3.1.31.1) further purification. Deoxynucleotides were from P-L Bio- and spleen exonuclease (EC 3.1.16.1). The digests were treated chemicals. [-32P]ATP was from Amersham, T4 polynucleotide with [V-3PJATP and T4 polynucleotide kinase (ATP:5'-dephos- kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransfer- phopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert ase, EC 2.7.1.78) was from P-L Biochemicals, and potato apyr- the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis- ase (ATP diphosphohydrolase, EC 3.6.1.5; grade I, 0.002 unit/ phosphates. These compounds were then separated on polyethyl- Ag) was from Sigma. Micrococcal endonuclease (EC 3.1.31.1; eneimine-cellulose thin layers in ammonium formate and am- grade VI, 0.21 unit/pkg; Sigma) and spleen exonuclease (EC monium sulfate solutions. Autoradiograms of the chromatograms 3.1.16.1; 0.002 unit/pg; Boehnnger Mannheim) were dialyzed obtained by this high-resolution procedure showed the presence against water at 40C for 15 hr. Calf thymus DNA was obtained ofnucleotides derived from chemically altered, as well as normal, DNA constituents. Maps firom DNA exposed to any of the chem- from Sigma. Polyethyleneimine (PEI)-cellulose thin layers (25 icals used exhibited a spot pattern typical for the particular chem- cm x 20 cm), prepared as described (14), were predeveloped ical. This method detected a single adduct in 105 DNA nucleotides in water for 12-15 hr (14) prior to use. XAR-5 films (Eastman without requiring that the compound under investigation be ra- Kodak) and Lightning Plus intensifying screens (Du Pont) were dioactive and thus provides a useful test to screen chemicals for used for autoradiography. DNA concentration was estimated their capacity to damage DNA by covalent binding. spectrophotometrically at neutral pH using 20 ANO units/mg for native DNA as standard (15). As shown by the Salmonella test established by Ames and co- Alkylation of DNA. Calf thymus DNA (25 jug) was allowed workers (1, 2) and by other tests (3-6), most carcinogens are to react with various chemicals as specified in Table 1. The either mutagens (i.e., electrophilic reactants that covalently treated DNA was purified by precipitation with alcohol (2.5 vol) bind to DNA bases) or premutagens (i.e., chemicals that at pH 5.0 or dialysis (40C, 15 hr) as indicated in Table 1. Control undergo metabolic conversion to such electrophilic compounds) incubations were performed in the absence of test chemicals. (7-10). Damage to DNA by chemicals is likely to be a major Digestion, 32P Labeling, and Mapping of Labeled Digests. cause ofcancer (2, 7, 8) and may also play a role in otherdiseases Treated or control DNA was quantitatively digested to deoxy- (2). It is therefore important to identify DNA-reactive chemicals nucleoside 3'-monophosphates by incubating 1 jug of DNA in the human environment. Surprisingly, no sensitive and gen- (=3 nmol of DNA-P) at 37°C for 2 hr with 2 ,ug each of micro- erally applicable test has been developed to detect directly the coccal endonuclease and spleen exonuclease in 10 p1 of20 mM presence of chemically altered bases in DNA. Most assays are sodium succinate/8 mM CaCl2, pH 6.0. For 32P labeling, the for the binding ofradioactively labeled or fluorescent test com- digest was diluted 1:10 and a 1-,ul aliquot containing %30 pmol pounds to DNA and are thus not generally applicable to screen- of deoxynucleoside 3'-monophosphates was added to 10 pA of ing large numbers of chemicals (4). A method for the analysis 10 p.M [Y-32P]ATP (100 Ci/mmol; 1 Ci = 3.7 X 10'0becquerels)/ of nonradioactive modified constituents in RNA that involves 40 mM N,N-bis(2-hydroxyethyl)glycine-NaOH, pH 9.0/10 mM digestion ofthe RNA to monomers, incorporation ofradioactive MgClJ/1o mM dithiothreitoV0. 1 mM spermidine/T4 polynu- label into the latter, thin-layer chromatography, autoradio- cleotide kinase (0.25 unit/1A). Incubation was at 37C for 1 hr. graphy or fluorography, and assay for radioactivity has been The 32P-labeled reaction mixture was further incubated with 3 reported (11). We have recently developed aformally analogous Al of apyrase (5 milliunits/jl) at 370C for 30 min to convert method for the quantitative analysis of normal DNA constitu- unreacted [y32P]ATP to 3'Pi. Three microliters of0.2 M sodium ents (12). Here we report an extension ofthis method to detect tungstate was added to the reaction mixture. Then, 1 p.1 of a the presence oftraces ofchemically altered constituents in DNA mixture ofauthentic dpGp, dpAp, dpCp, and dpTp (2 pLg each) damaged by mutagens. Part of this work has been communi- and 2 p1 ofthe reaction mixture (3.5 pmol of[32P]deoxynucleoside in form 3',5'-bisphosphates) were applied to a PEI-cellulose sheet 1.5 cated preliminary (13). cm from the lower edge and 2.5 cm from the left-hand edge. was with water to the then The publication costs ofthis article were defrayed in part bypage charge The chromatogram developed origin, payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: PEI, polyethyleneimine. 6126 Biochemistry: Randerath et aL Proc. Natd Acad. Sci. USA 78 (1981) 6127 Table 1. Conditions for DNA treatment ..f Incu- DNA Concen- bation purifi- tration, DNA-P, time at cation Mutagen mM mM Buffer* 370C, hr methodt None (control) 0 0.6 A 2 P N-Methyl-N- nitrosourea 80 3 A 2 P a. Control b. N-Methyl-N- c. Dimethyl sulfate Dimethyl sulfate 45 1 A 0.5 D nOirOsUrre Formaldehydet 370 1.5 B 4 D ,&Propiolactone 260 3 A 12 P Propylene oxide 200 3 C 12 P Streptozotocin 40 0.6 A 1.5 P Nitrogen mustard§ 43 0.6 B 5 D 1,3-Bis- (2-chloroethyl)- 1-nitrosourea 40 0.6 A 1.5 P * A, 0.2 M Tris HCl, pH 8.0; B, 0.2 M K phosphate, pH 8.5; C, 1.5 M NaOAc, pH 5.8. d. Formaldehyde e. 0- tP, alcohol precipitation; D, dialysis. t DNA was denatured by heating at 1000C for 10 min before exposure to formaldehyde. § This chemical contained NaCl (concentration in the reaction mix- ture, 1.1 M). with 4 M lithium formate/7.5 M urea, pH 4.0 (prepared by ti- tration of a formic acid/urea mixture with lithium hydroxide) to 5 cm above the origin. The sheet was soaked in 300 ml of methanol for 10 min, dried in a current of cool air, and cut 1 cm above the origin. The lower part, containing mainly 32P, (retained at the origin as a tungstate complex), was discarded. g. Streptozotocin h. Nitrogen i. ,'3-Bis(2-chloro- The upper portion was developed with 0.04 M ammonium for- mustard eff)yO-I-nitrosourea mate, pH 3.5 (prepared by titration of formic acid with am- monium hydroxide) to 3 cm from the cut edge, then, without intermediate with 0.7 M FIG. 1. Pmaps ofdigestsofcontrolcalfthymusDNA(a)andcalf drying, ammonium formate, pH 3.5, thymus DNA treated with the indicated electrophilic chemical (b-i). to 6 cm on a Whatman 1 wick that had been attached 21-22 cm DNA samples were digested to deoxynucleoside 3'-monophosphates, from the bottom edge by stapling and folded back. After chro- which were 32P labeled, and the resulting 3',5'-bisphosphates were matography, the layer was dried in a current ofcool air followed mapped on PEI-cellulose thin layers. [y.32P]ATP had a specific activity by warm air (5 min each). The sheet was soaked in 300 ml of0.01 of 100 Ci/mmol. The labeled DNA digests [total radioactivity, 1.2 AuCi; M Tris base in water for 10 min, then in 300 ml of water for 5 3.5 pmol (0.35 MCi) of [32P]deoxynucleoside 3',5'-bisphosphates] were min and dried. in the second chromatographed on anion-exchange thin-layer sheets of PEI-cellu- Chromatography dimension was lose in formate (bottom to top) and sulfate (leftto right) solutions. Auto- performed with 0.01 M Tris HCl, pH 8.0, to the origin, then radiography was at -70°C for 7-9 hr, except for samples a and i, which with 0.1 M ammonium sulfate/0.01 M Tris HCl, pH 8.0, to 7 were exposed for 12 and 16 hr, respectively. Spots of dpGp (G), dpAp cm on a Whatman 1 wick attached to the top ofthe sheet.