[1] DETERMINING OXIDATIVE DNA DAMAGE BY GC/MS 3

[1] Chemical Determinat ion o f Oxidative D N A Damage by Gas Chromatography-Mass Spectrometry

B y MIRAL DIZDAROGLU

Introduction

Oxidative DNA damage produced by free radicals or other DNA- damaging agents has been implicated to play a role in mutagenesis, carcino- genesis, reproductive cell death, and aging.l Oxygen-derived species such as superoxide radical (O2-) and H202 are generated in all aerobic cells. 1,2 Excess generation of these species by endogenous sources or exogenous sources (e.g., redox-cyclic drugs, ionizing radiation) may cause damage to cellular DNA by a variety of mechanisms): The toxicity of these species in vivo, however, is thought to result from their metal ion-catalyzed conversion to the highly reactive hydroxyl radical (-OH). l Ionizing radia- tion can also produce .OH in cells and tissues by interacting with cellular water. 3 Hydroxyl radical causes formation of a number of modified bases and sugars in DNA, and DNA-protein cross-links in nucleoprotein) -7 A number of these lesions are also produced by nonradical mechanisms.8-15 For understanding of the role of oxidative DNA damage in carcinogenesis

i B. Halliwell and J. M. C. Gutteridge, "Free Radicals in Biology and Medicine," 2nd ed. Oxford Univ. Press (Clarendon), Oxford, 1989.

2 I. Fridovich, Arch. Biochem. Biophys. 247, 1 (1986). 3 C. von Sonntag, "The Chemical Basis of Radiation Biology," pp. 116-166, 221-294.

Taylor & Francis, London, 1987. 4 B. Halliwell and O. I. Aruoma, FEBS Lett. 281, 9 (1991). 5 N. L. Oleinick, S. Chiu, N. Ramakrishnan, and L. Xue, Br. J. Cancer 55, Suppl. 8,

135 (1987). 6 M. Dizdaroglu, Free Radical Biol. Med. 10, 225 (1991). 7 K. Frenkel, Pharmacol. Ther. 53, 127 (1992). 8 M. Dizdaroglu, E. Holwitt, M. P. Hagan, and W. F. Blakely, Biochem. J. 235, 531 (1986). 9 R. A. Floyd, M. S. West, K. L. Eneff, and J. E. Schneider, Arch. Biochem. Biophys.

273, 106 (1989). l0 S. Steenken, Chem. Rev. 89, 503 (1989). ii S. A. Akman, J. H. Doroshow, and M. Dizdaroglu, Arch. Biochem. Biophys. 282, 202

(1990). 12 D. J. Deeble, M. N. Schuchmann, S. Steenken, and C. von Sonntag, J. Phys. Chem. 94,

8186 (1990). J3 D. Angelov, M. Berger, J. Cadet, N. Getoff, E. Keskinova, and S. Solar, Radiat. Phys.

Chem. 37, 717 (1991). 14 S. Boiteux, E. Gajewski, J. Laval, and M. Dizdaroglu, Biochemistry 31, 106 (1992). i5 H. Sies and C. F. M. Menck, Mutat. Res. 275, 367 (1992).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 234 All rights of reproduction in any form reserved.

4 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [ l ]

and other biological processes, it is essential to chemically characterize and quantify DNA lesions. In this chapter, we describe characterization and quantification of oxidative DNA damage by the technique of gas chromatography-mass spectrometry (GC/MS).

Materials and Methods

Reagents and enzymes used in this methodology are available commer- cially from a number of suppliers.16

Reference Compounds. Isobarbituric acid (5-hydroxyuracil), 5,6-dihy- drothymine, 6-azathymine, 8-azaadenine, 5,6-dihydrouracil, alloxan, 5-(hydroxymethyl)uracil, 4,6-diamino-5-formamidopyrimidine, 8-bromo- adenine, isoguanine (2-hydroxyadenine), 2,5,6-triamino-4-hydroxypyri- midine, and xanthine-l,3-15N2 are available from Sigma Chemical Com- pany (St. Louis, MO), and 8-hydroxyguanine from Schweizerhall Inc. (formerly Chemical Dynamics Corporation) (Piscataway, N J). Thymine- c~,ot, ot,6-2H4 is available from Merck & Co. Inc./Isotopes (St. Louis, MO).

8-Hydroxyadenine and 2,6-diamino-4-hydroxy-5-formamidopyrimi- dine are synthesized by treatment of 8-bromoadenine and 2,5,6-triamino- 4-hydroxypyrimidine with formic acid, respectively. 17,18 cis-Thymine gly- col is obtained by treatment of thymine with osmium tetroxide. 8 5-Hydroxy-5-methylhydantoin is obtained by the reaction between pyru- vic acid and u r e a . 19 Isodialuric acid (5,6-dihydroxyuracil) is synthesized by oxidation of isobarbituric acid with bromine. E° 5-Hydroxyhydantoin is obtained by treatment of alloxan with formic a c i d . E1 These and other reference compounds, and their stable isotope-labeled analogs, which are dealt with in this chapter, are available on a custom-synthesis basis from the Chemical Synthesis and Analysis Laboratory of Program Re- sources Inc./Dyncorp, National Cancer Institute-FCRD (Frederick, MD). The following stable isotope-containing analogs of modified DNA bases have also become available: 5,6-dihydrothymine-l,3JSNE-2J3C, 5,6- dihydrouracil-l ,3JS NE-E J3C, 5-hydroxy-5-methylhydantoin-l ,3JS NE-E J3 C,

16 Certain commercial equipment or materials are identified in this chapter in order to specify adequately the experimental procedure. Such identification does not imply recommenda- tion or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

17 M. Dizdaroglu and D. S. Bergtold, Anal. Biochem. 156, 182 (1986). 18 L. F. Cavalieri and A. Bendich, J. Am. Chem. Soc. 72, 2587 (1950). ~9 S. Murahashi, H. Yuki, K. Kosai, and F. Doura, Bull. Chem. Soc. Jpn. 39, 1559 (1966). 2o R. Behrend and O. Roosen, Justus Liebig's Ann. Chem. 251, 235 (1889). 21 M. Dizdaroglu, FEBS Lett. 315, 1 (1993).

[1] DETERMINING OXIDATIVE D N A DAMAGE BY G C / M S 5

alloxan-1,3-15N2-2,4-13C2, 5-hydroxyhydantoin-1,3A5N2-2,433C 2, 5-hy- droxyuracil-l,3JSN2-2J3C , 5-(hydroxymethyl)uracil-2,433C2-a,a-2H 2 , 5-hydroxycytosine-l,3A5N2-2A3C, cis-thymine glycol-a,a,a,6-2H4, 5,6- dihydroxyuracil-I,3J5N2-2J3C (isodialuric acid-l,3A5N2-233C), 4,6-dia- mino-5-formamidop yrimidine-l ,3-15 N2-2-~3 C-(5-f ormamidoA5 N,2 H ) , 8-hy- droxyadenine-l,3,7-tSN3-2,8J3C2, 2,6-diamino-4-hydroxy-5-formamido- pyrimidine-l,3J5N2-(5-amino-15N)-2A3C, and 8-hydroxyguanine-l,3JSN 2- (2-aminoA5N)-233C. The synthesis of 5-(hydroxymethyl)uracil-2-13C-5 - 2H2-6-2H has also been reported elsewhere. 22

Most compounds listed above are soluble in water. 8-Hydroxyguanine and 8-hydroxyguanine-l,3-t5N2-(2-amino35N)-233C are not completely soluble. However, complete solubility can be obtained by increasing the pH of the solutions to 9.5 with dilute NaOH and then stirring the solutions for several hours at room temperature.

Hydrolysis

The preparation of DNA or nucleoprotein samples for analysis by GC/MS involves hydrolysis followed by derivatization. DNA can be hy- drolyzed by either acidic hydrolysis or enzymatic hydrolysis.

Acidic Hydrolysis. Acidic hydrolysis cleaves the glycosidic bonds be- tween bases and sugar moieties in DNA, releasing intact and modified bases. Formic acid is well suited for hydrolysis of DNA. z3-25 In the case of DNA-protein cross-links, nucleoprotein is hydrolyzed by the standard method of protein hydrolysis using 6 M HC1. By this procedure, peptide bonds in proteins as well as glycosidic bonds in DNA are cleaved to release DNA base-amino acid c r o s s - l i n k s . 26'27

The stability of numerous pyrimidine- and purine-derived modified DNA bases and their release from DNA have been studied under various conditions of formic acid hydrolysis, because this information is important for the assessment of the accuracy of the DNA damage measurement. 25,28 It was found that most of the modified bases are stable under all studied conditions of hydrolysis, and only a few undergo partial destruction, de- pending on the concentration of formic acid. 25 Furthermore, the possibility that some of the modified bases may be formed by acidic treatment in

22 Z. Djuric, D. A. Luongo, and D. A. Harper, Chem. Res. Toxicol. 4, 687 (1991). 23 G. R. Wyatt and S. S. Cohen, Biochem. J. 55, 774 (1953). 24 M. Dizdaroglu, Anal. Biochem. 144, 593 (1985). z5 Z. Nackerdien, R. Olinski, and M. Dizdaroglu, Free Radical Res. Commun. 16, 259 (1992). 26 E. Gajewski, A. F. Fuciarelli, and M. Dizdaroglu, Int. J. Radiat. Biol. 54, 445 (1988). 27 M. Dizdaroglu, E. Gajewski, P. Reddy, and S. A. Margolis, Biochemistry 28, 3625 (1989). 28 A. F. Fuciarelli, B. J. Wegher, E. Gajewski, M. Dizdaroglu, and W. F. Blakely, Radiat.

Res. 119, 219 (1989).

6 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [1]

DNA from corresponding intact bases has been investigated. The results indicated that formic acid caused no significant formation of modified bases under the conditions used. As a conclusion of these studies, formic acid at a concentration of 60% (v/v) has been found to be optimal for DNA hydrolysis. 25

The following procedure is used for DNA hydrolysis. An aliquot of DNA (10 to 100/zg) is treated with 0.5 ml of formic acid (60%) in evacuated and sealed tubes at 140 ° for 30 min. The sample is then transferred to a vial, frozen in liquid nitrogen, and lyophilized. If chromatin instead of pure DNA is to be hydrolyzed for detection of modified bases, the same hydrolysis procedure is followed. It should be pointed out that formic acid hydrolysis causes deamination and dehydration of cytosine-derived products as follows: cytosine glycol yields a mixture of 5-hydroxycytosine and 5-hydroxyuracil, the former by dehydration and the latter by dehydra- tion and deamination. 8 5,6-Dihydrocytosine, 5-hydroxy-6-hydrocytosine and 5,6-dihydroxycytosine deaminate to give 5,6-dihydrouracil, 5-hy- droxy-6-hydrouracil and 5,6-dihydroxyuracil, respectively. 5-Hydroxy- hydantoin, which has been identified in the past as a product of cytosine, 29 results from acid-induced decarboxylation of alloxan. 21

For detection of DNA-protein cross-links, nucleoprotein containing 100 ~g DNA is hydrolyzed with 0.5 ml of 6 M HCI in evacuated and sealed tubes at 120 ° for 6 hr. Subsequently, the sample is transferred into a vial, frozen in liquid nitrogen and lyophilized.

Enzymatic Hydrolysis. This type of hydrolysis has been discussed elsewhere in detail. 3° The following procedure can be used to hydrolyze DNA to nucleosides. An aliquot (0.1 mg) of DNA is incubated in 0.5 ml of 10 mM Tris-HCl buffer, pH 8.5 (containing 2 mM MgC12), with deoxyribonuclease I (100 units), spleen exonuclease (0.01 unit), snake venom exonuclease (0.5 units), and alkaline phosphatase (10 units) at 37 ° for 24 hr. The sample is then transferred to a vial, frozen in liquid nitrogen, and lyophilized.

The drawback of enzymatic hydrolysis is that the products of the 2'-deoxycytidine moiety in DNA cannot be readily analyzed by GC/MS because of the poor gas chromatographic properties of cytidine deriva- tives. 31 On the other hand, generally less volatile trimethylsilyl [(CH3)3Si] derivatives of modified purine 2'-deoxynucleosides can be analyzed suc- cessfully by GC/MS.31'32 Deamination of 2'-deoxyadenosine products may

29 M. Polverelli and R. Troule, Z. Naturforsch., C: Biosci. 29C, 12 (1974). 3o p. F. Crain, this series, Vol. 193, p. 782. 3z M. Dizdaroglu, J. Chromatogr. 367, 357 0986). 32 M.-L. Dirksen, W. F. Blakely, E. Holwitt, and M. Dizdaroglu, Int. J. Radiat. Biol. 54,

195 (1988).

[1] DETERMINING OXIDATIVE DNA DAMAGE BY GC/MS 7

occur during enzymatic hydrolysis owing to contaminating deaminase activity in the enzymes. Removal of excess salt from the hydrolyzates and removal of deaminases from the enzymes may prevent problems associated with analysis of 2'-deoxycytidine products and deamination of 2'-deoxyadenosine products. 3°'33

Derivatization

DNA bases, nucleosides, and DNA base-amino acid cross-links are not sufficiently volatile for gas chromatography, and thus must be con- verted to volatile derivatives. For this purpose, trimethylsilylation is the mode of derivatization most frequently used. 34 (tert)-Butyldimethylsilyla- tion is also u s e d . 33'35'36 The following procedure can be used for trimethyl- silylation. Lyophilized hydrolyzates of DNA or nucleoprotein samples containing 0.01-0.1 mg of DNA are heated with 0.1 ml of a mixture of bis(trimethylsilyl)trifluoroacetamide (containing I% trimethylchlorosi- lane) and acetonitrile (4 : 1, v/v) at 140 ° for 30 rain in polytetrafluoroethyl- ene-capped vials (sealed under dry nitrogen). The amounts of the reagents can be modified according to the amount of DNA. After derivatization, samples are cooled to room temperature. Without any further treatment, an aliquot (e.g., l tzl) of each derivatized sample is injected into the injec- tion port of the gas chromatograph.

I n s t r u m e n t a t i o n

A GC/MS instrument equipped with a capillary inlet system and a computer data system can be used. Data presented and reviewed here were obtained on commercial quadrupole mass spectrometers. Fused- silica capillary columns are used for separation of derivatized hydrolyzates of DNA or nucleoprotein. These types of columns provide high inertness, excellent separation efficiency, and measurement of high sensitivity. Fused-silica capillary columns coated with cross-linked 5% phenyl methyl- silicone gum phase appear to be best for the p u r p o s e . 24'37 Column length may vary depending on the type of analysis. A column 12.5 m in length (0.2 mm internal diameter, 0.33 tzm film thickness) is generally used for analysis of derivatized bases. A shorter column (e.g., 8 m, 0.2 mm internal

33 p. F. Crain, Mass Spectrom. Rev, 9, 505 (1990). 34 K. H. Schram, this series, Vol. 193, p. 791. 35 M. A. Quilliam and J. B. Westmore, Anal. Chem. 50, 59 (1978). 36 M. Dizdaroglu, BioTechniques 4, 536 (1986). 37 M. Dizdaroglu, J. Chromatogr. 295, 103 (1984).

8 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [1]

diameter, 0.11 /~m film thickness) serves best for analysis of derivatized DNA base-amino acid cross-links.

Helium (ultrahigh purity) is used as the carrier gas. The split mode of injection is used to avoid overloading the column. The split ratio (i.e., ratio of the carrier gas flow through the splitter vent to carrier gas flow through the column) is adjusted according to the DNA amount in samples. An aliquot of DNA hydrolyzates corresponding to approximately 0.1 to 0.4 ~g of DNA on the GC column after splitting of the injected sample is generally sufficient. The injection port of the gas chromatograph and the GC/MS interface are kept at 250 °. The temperature of the ion source of the mass spectrometer is usually kept at around 200 ° . Some instruments may permit variation of this temperature zone. The glass liner in the injection port of the gas chromatograph is filled with silanized glass wool, which allows homogeneous vaporization of injected samples. Electron- ionization (El) mode at 70 eV has been used to record mass spectra and to perform selected-ion monitoring in studies presented or reviewed here.

Gas Chromatography-Mass Spectrometry

Free Bases. Gas chromatography on a fused-silica capillary column (usually 12.5 m long) permits separation of (CH3)3Si derivatives of a large number of modified bases from one another and from the four intact DNA bases in a single analysis. 24 Compounds eluting from the GC column are ionized in the ion source and then analyzed by the mass analyzer of the mass spectrometer) 8 Electron-ionization mass spectra of (CH3)3Si derivatives of modified DNA bases provide considerable structural detail that can be used for unequivocal identification. These mass spectra are characterized by an intense molecular ion (M "+ ion), an intense (M - 15) ÷ ion, and other characteristic ions , 24'37 as are those of the intact bases, a9'4° The (M - 15) + ion results from the loss of a methyl radical from the M .+ ion. 39'4° In some instances, an intense (M - l) ÷ ion resulting from loss of an H atom from the M .+ ion also appears in the mass spectra . 24'37

Nucleosides. Trimethylsilyl derivatives of modified 2'-deoxynucle- osides follow the same fragmentation patterns as those of other nucle-

38 j . T. Watson, "Introduction to Mass Spectrometry," Chapters 1 and 3. Raven, New York, 1985.

39 E. White, V. P. M. Krueger, and J. A. McCIoskey, J. Org. Chem. 37, 430 (1972). 40 j . A. McCloskey, in "Basic Principles in Nucleic Acid Chemistry" (P. O. P. Ts'o, ed.),

Vol. l, p. 209. Academic Press, New York, 1974.

[1] DETERMINING OXIDATIVE D N A DAMAGE BY GC/MS 9

osides/1,42 Prominent ions are the (base + H) + ion [(B + 1) + ion] and the (base + H - CH3) + ion, whereas the M .+ and the (M - 15) + ions are of low intensity. 31 For example, in the mass spectra of (CH3)3Si deriva- tives of 8-hydroxy-2'-deoxyguanosine and 8-hydroxy-2'-deoxyadenosine, the (B + 1) + ion appears as the most prominent ion (100% relative inten- sity) owing to stabilization through an electron-donating substituent at C-8 of the purine ring. Trimethylsilyl derivatives of 8,5'-cyclopurine 2'-deoxynucleosides provide partly different fragmentation patterns from those of other nucleosides. 32'43 These spectra are characterized by promi- nent ions containing the base plus portions of the sugar moiety and by an intense M "+ ion, most likely due to stabilization by the increased number of rings in the molecule. 44

DNA Base-Amino Acid Cross-links. Mass spectra of (CH3)3Si deriva- tives of DNA base-amino acid cross-links contain M .+ and (M - 15) + ions and other characteristic ions resulting from typical fragmentations of base and amino acid moieties, z6'~7'37 For example, the most prominent ion (m/z 448) in the mass spectrum of the (CH3)3Si derivatives of the thymine-tyrosine cross-link results from cleavage of the bond between the a and/3 carbons of the tyrosine moiety accompanied by an H atom transfer [(M - 218 + 1) + ion]. The high abundance of this ion is due to resonance stabilization through the aromatic ring. This cleavage, which is typical of (CH3)3Si derivatives of amino acids, also accounts for the intense m/z 218 ion when the charge is retained on the a carbon without an H atom transfer. In the case of DNA base-aliphatic amino acid cross- links, these fragmentations also occur. However, an ion arising from loss of "CO~Si(CH3)3 from the M "+ ion generally appears as one of the most prominent ions in the mass spectra, whereas the abundance of the (M - 218 + 1) + ions depends on the aliphatic amino acid residue.

Figures 1 and 2 illustrate the structures of modified DNA bases and nucleosides and some DNA base-amino acid cross-links that can be mea- sured by the use of the GC/MS technique. These compounds are formed in DNA or nucleoprotein by free radicals or other agents causing oxidative damage. 6,45 DNA base-amino acid cross-links involving thymine and the amino acids glycine, alanine, valine, leucine, isoleucine, and threonine,

41 H. Pang, K. H. Schram, D. L. Smith, S. P. Gupta, L. B. Towsend, and J. A. McCloskey, J. Org. Chem. 47, 3923 (1982).

42 j. A. McCloskey, this series, Vol. 193, p. 825. 43 M. Dizdaroglu, Biochem. J. 238, 247 (1986). 44 F. W. McLafferty, "Interpretation of Mass Spectra." Univ. Sci. Books, Mill Valley,

California, 1980. 45 M. Dizdaroglu, Mutat. Res. 275, 331 (1992).

10 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [1]

,~= • It" =... =:

- r

o = ~! ==~o

o " }

0 0 0 ~" . ~

0 ~ .~

~ = " i . =

= % =

o z ~ :: 0=~-=

o -,- ~ o -~'~ .= = ,~o

0 ~" : / / Eta == ~ == =

z = 0 "1" -~.

z ~ = 0

= .d== =._~o = ~

0 Z .:

z : ~ 2 : o o z =. z =

t .

.=. ~;

"o

== ~. _ ~

~ - o o ~- - ~ ~ < ~

:~ ~ - - ~ / z ~._~

6

= o i ~ "~ ~- 0 ..Q ".'- e,I

• - ~== ~ - ~

. j .

[1] DETERMINING OXIDATIVE D N A DAMAGE BY GC/MS I 1

which are also measurable by this technique, are not illustrated in Fig. 2. It should be noted that the exact structure of the thymine-tyrosine cross- link has been determined. Structures ofthymine-lysine and cytosine-tyro- sine cross-links illustrated in Fig. 2 have been proposed on the basis of their mass spectral fragmentation patterns. 6

Measurement at Low Analyte Concentrations

Identification and quantification of components of a complex mixture at low concentrations are generally carried out using GC/MS with selected-ion monitoring ( S I M ) . 46 When using this mode for identification, knowledge of the mass spectrum and the retention time of the analyte is required. A number of characteristic ions of an analyte are monitored by the mass spectrometer during the time period in which the analyte elutes from the GC column. If the analyte is present in the mixture, signals of the monitored ions will line up at its expected retention time. Subsequently, a partial spectrum is obtained on the basis of the moni- tored ions and their relative abundances. This spectrum is then compared with that of the authentic compound for unequivocal identification. For this purpose, the mass spectrum of the authentic compound should be recorded under the same tuning conditions of the mass spectrometer as are used to perform the SIM. This is because differences in the relative abundances of ions may occur depending on the tuning conditions of the mass spectrometer. Retention times of analytes also play an import- ant role in reliable identification in addition to simultaneous measurement of masses because gas chromatography on fused-silica capillary columns permits measurement of retention times with great accuracy and pre- cision.

Quantification. The GC/MS-SIM technique also permits accurate quantification of components of a complex mixture.46 This is achieved by adding an aliquot of a suitable internal standard to aliquots of DNA samples at an early stage such as prior to hydrolysis. In mass spectrometry, a stable isotope-labeled analog of an analyte can be used as the internal standard. 46 This procedure is called isotope-dilution mass spectrometry. Because of the essentially same physical and chemical properties of the analyte and its analog, the procedure permits compensation for possible losses of the analyte during sample preparation and GC/MS analysis. If stable isotope-labeled analogs are not available, structurally similar compounds may be used as internal standards. 38 In the past, such com- pounds have been used for quantification of modified DNA bases. 6'z5'28

46 j. T. Watson, this series, Vol. 193, p. 86.

12 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [1]

o "i

=-4 |

o -r -r

Y ~

,. ~.g

• r r ~

0 ~

. , ~ ~ . _ ~ , , . , - ~ ~

g g g= _ ~

=° ~ .,:_-i, ~o (J--(J--Z co

< Z

- le

• ~ ! - z

o= , ~ o ~ - r L T ~

L~ {D

[1] DETERMINING OXIDATIVE D N A DAMAGE BY GC/MS 13

Recently, stable isotope-labeled analogs of modified DNA bases have become available (see Materials and Methods). 21,22

Mass spectral fragmentation patterns of stable isotope-labeled analogs are similar to those of corresponding unlabeled compounds. Masses of most ions in the mass spectra of labeled analogs are shifted to higher masses according to the isotope contents. 2]'22 Typical examples of mass spectra are illustrated in Fig. 3. Here are shown the mass spectra of (CH3)3Si derivatives of 4,6-diamino-5-formamidopyrimidine (Fig. 3A) and 4,6-diamino-5-formamidopyrimidine-tSN 3 ,13C,2H (Fig. 3B). In Fig. 3A, the M +- , the (M - 1) +, and the (M - 15) + ions appear at m/z 369, 368, and 354, respectively. The intense ion at m/z 280 results from the loss of • OSi(CH3)3 (89 Da) from the M .+ ion . 24 In Fig. 3B, the masses of these ions are shifted by 5 Da to 374, 373,359, and 285 Da, respectively. The ion at m/z 372 may result from the M +. ion by loss of an 2H atom located at the formyl group of the molecule. Ions at m/z 73 and 147 in Fig. 3A,B are commonly observed with (CH3)3Si derivatives. 39

Trimethylsilyl derivatives of modified DNA bases coelute with those of their ]3C- and ]SN-labeled analogs, indicating no isotope effect on elution behaviors of corresponding compounds. 2] However, if a labeled analog contains several 2H atoms such as thymine glycol-2H4, a slight resolution from the unlabeled material is observed, 21 indicating a well-known isotope

'ee]l~ A

40 147 19e

18~ 15~ 28~ 258 m/z

38 354

388 35~

8 8 5

I ~ 15~ 2~8 258 3~8 35~ m/z

FIG. 3. (A) Electron-ionization mass spectrum of the (CH3)3Si derivative of 4,6-diamino- 5-formamidopyrimidine; (B) EI mass spectrum of the (CH3)3Si derivative of 4,6-diamino-5- formamidopyrimidineJSNs,uC,2H. (From Dizdaroglu. 21)

14 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [1]

effect on the elution behavior of 2H-containing compounds. Figure 4 illus- trates superimposed selected ion-current profiles of three compounds and their labeled analogs. A discernible resolution of thymine glycol from its 2H-labeled analog is seen, whereas 2,6-diamino-4-hydroxy-5-formamido- pyrimidine and 8-hydroxyguanine coelute with their respective ~3C- and lSN-labeled analogs.

For quantification, calibration plots are obtained prior to GC/MS analy- sis for the response of the mass spectrometer to known quantities of both the analyte and its stable isotope-labeled analog as the internal standard. 46 For this purpose, mixtures containing known quantities of the analyte and analog are analyzed by GC/MS-SIM, and a number of prominent characteristic ions are monitored. The ratios of ion currents at selected masses are plotted as a function of the ratios of the molar amounts of an analyte and its analog. A linear relationship of the ratio of ion currents to the ratio of quantities should be obtained. Subsequently, quantities of analytes in a given mixture are calculated using the areas of the ion-current profiles of the monitored ions and the corresponding calibration plots. Examples of calibration plots are illustrated in Fig. 5. Although the data points were obtained by three independent measurements and contain standard deviations, 2~ the error bars are not discernable on the plots. This

A 10000"

s00o- m/z 262

8 0 0 0 "

7000"

6000"

5000"

4000 m/'z 259

30001 2000

6 . 4 0 6 . 7 0 Time (mln.)

B so00 m/z 446

5000

4000

m/z 442 3000

2000

l O 0 0

10.60 10.B5 T t m o (mln.)

C 90000~

80000 m/z 444

7 0 0 0 0

60000-

50000"

40000"

30000"

20000. m/z 440

10000'

1 . 9 12.2 Tlmo (mln.)

FIG. 4. Superimposed selected ion-current profiles (A) at m/z 259 [(CH3)3Si derivative of thymine glycol] and 262 [(CH3)3Si derivative of thymine glycol-2H4]; (B) at m/z 442 [(CH3)3Si derivative of 2,6-diamino-4-hydroxy-5-formamidopyrimidine] and 446 [(CH3)3Si derivative of 2,6-diamino-4-hydroxy-5-formamidopyrimidineJSN3,13C]; (C) at rn/z 440 [(CH3)3Si derivative of 8-hydroxyguanine] and 444 [(CH3)3Si derivative of 8-hydroxyguanine- 15N3,13C]. Profiles were obtained during the GC/MS-SIM analysis of a trimethylsilylated hydrolyzate of y-irradiated chromatin. (From Dizdaroglu. 2~)

[1] DETERMINING OXIDATIVE DNA DAMAGE BY GC/MS 15

2.0

g g

"~ 1.o _o

0 . 0 0 .0 0 , 5 1 .0 1 .5 2 . 0 2 .5

ratio of amounts

FI6.5. Calibration plots for ratios of ion currents at indicated masses versus ratios of molar amounts of (CH3)3Si derivatives of 8-hydroxyguanine and 8-hydroxyguanineJSN3,'C (Q, m/z 440/444), and (CH3)3Si derivatives of 4,6-diamino-5-formamidopyrimidine and 4,6- diamino-5-formamidopyrimidine-tSN3,'C,2H (A, m/z 354/359). (From Dizdaroglu. 2~)

is because standard deviations were equal to or less than 1%, indicating the precision of such measurements by GC/MS-SIM.

Selectivity and Sensitivity. The GC/MS-SIM technique not only per- mits high-sensitivity measurements but also provides high selectivity by virtue of monitoring a few selected ions that are characteristic of only one analyte in a complex mixture. This unique characteristic of mass spectrometry combined with the precise measurement of retention times by capillary gas chromatography makes unequivocal identification of ana- lytes possible. Sensitivities in the range of approximately 1 fmol per com- pound applied to the GC column, or in the range of 1-3 modified residues in 10 6 DNA bases, can be achieved. It should be pointed out that the level of sensitivity may depend on the GC/MS instrument and the type of column and other factors. Mass spectrometers equipped with a high- energy dynode electron multiplier may provide approximately 10- to 20- fold higher sensitivity than the sensitivity range mentioned above for modified DNA bases.

Conclusions

The GC/MS technique has been applied to a variety of in vitro and in vivo studies of oxidative DNA damage. Extensive reviews of the applica- tions can be found elsewhere. 6'45 The technique offers the sensitivity,

16 OXIDATIVE DAMAGE TO D N A AND D N A REPAIR [2]

selectivity, speed, and versatility to solve a wide range of important mea- surement problems in terms of DNA base and sugar damage as well as DNA-protein cross-links. It also permits one to study enzymatic repair of DNA damage. 14,47 It appears that the GC/MS technique will continue to find a major role in studies of oxidative DNA damage and its repair in foreseeable future.

Acknowledgment

Work in the author's laboratory is supported in part by the Office of Health and Environ- mental Research, Office of Energy Research, U.S. Department of Energy, Washington, D.C.

47 M. Dizdaroglu, J. Laval, and S. Boiteux, Biochemistry 32, 12105 (1993).

[2] A s s a y s o f O x i d a t i v e D N A D a m a g e B i o m a r k e r s 8 - O x o - 2 ' - d e o x y g u a n o s i n e a n d 8 - O x o g u a n i n e in N u c l e a r

D N A a n d Bio logica l F l u i d s b y H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y wi th E l e c t r o c h e m i c a l D e t e c t i o n

By M A R K K. S H I G E N A G A , E L I A S N. A B O U J A O U D E ,

QIN CHEN, and BRUCE N. AMES

Introduction

Reactive oxygen species, which are generated as by-products of cellular metabolism, inflammation, ionizing radiation, and various xeno- biotic treatments, 1 produce lesions in DNA that may result in mutations. The postulated importance of oxidative DNA damage in aging, cancer, and other age-related degenerative processes has prompted the develop- ment of methods that meet the following criteria: (1) ability to measure oxidative DNA damage with high sensitivity and selectivity in biological samples; (2) ability to survey this damage in a wide array of sample types such as DNA isolated from various organs and cultured cells as well as the excised repair products in biological fluids such as urine

i B. Halliwell and J. M. Gutteridge, " F r e e Radicals in Biology and Medic ine ," 2nd ed. Oxford Univ. Press (Clarendon), Oxford, 1989.

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