ARCHIVES

Vol.

OF BIOCHEMISTRY

285, No. 2, March,

AND

BIOPHYSICS

pp. 317-324,

1991

Damage to the DNA Bases in Mammalian Chromatin Hydrogen Peroxide in the Presence of Ferric and Cupric Ions Miral

Dizdaroglu,*,’

Govind

Rae,?

Barry

Halliwell,$

and Ewa

by

Gajewski*

*Center for Chemical Technology, National Institute of Standards and Technology, Gaithersburg, Maryland 20899; jChemica1 and Biochemical Engineering, University of Maryland Baltimore County and Medical Biotechnology Center, Maryland Biotechnology Institute, Baltimore, Maryland 21228; and $Biochemistry Department, University of London King’s College, Strand Campus, London WC2R 2LS, United Kingdom Received

August

6, 1990, and in revised

form

November

1, 1990

Modification of DNA bases in mammalian chromatin upon treatment with hydrogen peroxide in the presence of ferric and cupric ions was studied. Ten DNA base products in mammalian chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring after hydrolysis of chromatin and trimethylsilylation of hydrolysates. This technique permitted the analysis of modified DNA bases in chromatin without the necessity of isolation of DNA from chromatin first. Modified bases identified were typical hydroxyl radical-induced products of DNA, indicating the involvement of hydroxyl radical in their formation. This was also confirmed by inhibition of product formation by typical scavengers of hydroxyl radical. The inhibition of product formation was much more prominent in the presence of chelated ions than unchelated ions, indicating a possible site-specific formation of hydroxyl radical when metal ions are bound to chromatin. Hydrogen peroxide in the presence of cupric ions caused more DNA damage than in the presence of ferric ions. Chelation of cupric ions caused a marked inhibition in product formation. By contrast, DNA was damaged more extensively in the presence of chelated ferric ions than in the presence of unchelated ferric ions. The presence of ascorbic acid generally increased the yields of the products, indicating increased production of hydroxyl radical by reduction of metal ions by ascorbic acid. Superoxide dismutase afforded partial inhibition of product formation only in the case of chelated iron ions. The yields of the modified bases in chromatin were lower than those observed with calf thymus DNA under the same conditions. 0 1991

Academic

Pm-s,

Inc.

1 To whom correspondence of Standards and Technology, FAX: (301) 330-3447.

should be addressed at National Institute Bldg. 222/B348, Gaithersburg, MD 20899.

0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Superoxide radical (0;) and Hz02 are produced in mammalian cells during aerobic metabolism (1,2). Excess generation of these species in viuo may result in damage to DNA. Thus oxygen-derived species may be mutagenic and carcinogenic (3-6). However, neither 0; nor Hz02 under physiological conditions appear to produce DNA strand breakage (7-9) or modification of DNA bases (10). Thus much of the toxicity of 0, and H202 in uiuo is thought to arise from their metal ion-catalyzed conversion into highly reactive hydroxyl radical (‘OH) (8, 11, 12). The necessary metal ions might exist bound to DNA in uivo, or the oxidative stress might cause their liberation from intracellular storage sites with subsequent binding to DNA (12). Evidence indicates that iron ions can lead to formation of ‘OH from 0, and Hz02 (12). Indeed, DNA base modifications that typically result from exposure of DNA to ionizing radiation in aqueous solution, a known source of ‘OH, are also formed in DNA treated with O;producing systems and with H202 in the presence of iron ions (10, 13, 14). HzOz with copper ions also produces extensive DNA strand breakage (15, 16), and causes formation of typical ‘OH-induced base products in DNA (17). Formation of ‘OH in the reaction of copper ions with HzOa has been suggested (15,17-21). However, some researchers have disputed the formation of ‘OH in copper ion/ HzOz systems (16,22,23). DNA strand breakage and formation of DNA base products by copper ion/HzOz has been shown to be more extensive than that by iron ion/ HzOz under similar experimental conditions (17, 20, 24). Hydroxyl radical produces a large number of sugar and base products in DNA, as well as DNA-protein crosslinks in nucleoprotein [for reviews see Refs. (25, 26)]. Modifications produced in cellular DNA may be repaired by cellular repair processes; however, if they are not re317

318

DIZDAROGLU

paired, they may have detrimental biological consequences (5, 27). Understanding of the biological consequences of free radical-induced DNA damage depends on the chemical characterization of the DNA lesions and the measurement of their quantities under various conditions of free radical formation. In the present work, we investigated the modification of DNA bases in mammalian chromatin in aqueous suspension exposed to HzOz in the presence of chelated or unchelated ferric or cupric ions. The technique of gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM)2 was used for this purpose. This technique permits the direct analysis of free radicalinduced products of all four DNA bases in chromatin without the necessity of first isolating DNA from chromatin (28). The objective of this work was to see whether the typical ‘OH-induced base products found in DNA in aqueous solution are also produced in mammalian chromatin exposed to H202 in aqueous suspension in the presence of metal ions. A further objective was to determine the differences between the effects of Fe3’ and Cu2+ on the types and amounts of DNA base products in chromatin, and to examine the effect of chelation of these ions. In living cells, DNA is not free but complexed with histones to form chromatin [for a review see Ref. (29)]. Histones that are closely associated with DNA in nucleosomes may also react with free radicals, and DNA bases may participate in formation of DNA-protein crosslinks in chromatin (26,30). For these reasons, we believe that mammalian chromatin represents a more biologically relevant model system than DNA alone in aqueous solution, and that it is important to study free radical damage to mammalian chromatin under various conditions of free radical production. MATERIALS

AND

METHODS

Moterials.3 Histones Hl, H2A, H2B, H3, and H4 were purchased from Boehringer Mannheim. Reagents for electrophoresis and Chelex 100 resin (200-400 mesh, sodium form) were obtained from Bio-Rad. Nitrilotriacetic acid (NTA), EDTA, dimethylsulfoxide (DMSO), mannitol, ascorbic acid (asc), 5-hydroxymethyluracil (B-OHMeUra), iso-

’ Abbreviations used: BSTFA, bis(trimethylsilyl)trifluoroacetamide; NTA, nitrilotriacetic acid; SOD, copper-zinc superoxide dismutase; DMSO, dimethylsulfoxide; GC/MS-SIM, gas chromatography/mass spectrometry with selected-ion monitoring; 5-OH-5-MeHyd, 5-hydroxy5methylhydantoin; 5-OH-Hyd, 5-hydroxyhydantoin; Cyt glycol, cytosine glycol; 5-OHMeUra, 5hydroxymethyluracik Thy glycol, thymine glycol; 5,6-diOH-Cyt, 5,6dihydroxycytosine; FapyAde, 4,6-diamino-5formamidopyrimidine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8-OH-Ade, 8-hydroxyadenine; 8-OH-Gua, 8-hydroxyguanine; chr, chromatin; asc, ascorbic acid. s Certain commercial equipment or materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation 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.

ET

AL.

barbituric acid (5-hydroxyuracil), 4,6-diamino-5formamidopyrimidine (FapyAde), 6-azathymine, 8-azaadenine, and copper-zinc superoxide dismutase (SOD) were purchased from Sigma Chemical Co. Units of SOD were as defined by the cytochrome c assay (31). 2-Amino-6,8-dihydroxypurine [8-hydroxyguanine (8-OH-Gua)] was from Chemical Dynamics Corp. Dialuric acid (5,6dihydroxyuracil) was purchased from American Tokyo Kasei, Inc. Synthesis of other base products was described elsewhere (32,33). Dialysis membranes with a molecular weight cutoff of 3500 were purchased from Fisher Scientific Co. Formic acid was obtained from Mallinckrodt. Cell culture and isolatian of chromatin. The cells used for chromatin isolation were Spa/O-derived murine hybridomas. The cell line has been designated HyHEL-10 and produces IgG antibodies against hen eggwhite lysozyme (courtesy of Dr. S. J. Smith-Gill, National Cancer Institute, Bethesda, MD). Cell culture and isolation of chromatin were as described previously (28). Chromatin was dialyzed extensively against 1 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA to remove any metal ions, followed by extensive dialysis against phosphate buffer (pH 7.4) treated with Chelex resin. All operations were carried out at 4’C. After dialysis, chromatin was homogenized briefly with a few strokes in a glass homogenizer. Characterization of chromatin. Chromatin was characterized as described previously (28). The ratio of the amount of protein to that of DNA was 1.6 (w/w). The RNA content of chromatin was 65% of the amount of DNA. The chromatin exhibited the following spectral characteristics: A168/A280 = 1.51; Azss/Az30 = 0.98; A268/Aam = 9.4; A(maximum)/A(minimum) = 1.38. The protein components of chromatin were analyzed by gel electrophoresis essentially as described by Laemmli (34), with a modification in the ratio of acrylamide to bis(acrylamide). Separating gel (18%) and stacking gel (4%) were prepared from a stock solution of 29.8% acrylamide and 0.2% bis(acrylamide). Electrophoresis was carried out in a 15 X 18-cm slab gel at 40 mA for 2 h. Gels were stained with Coomassie blue. Treatment of chromatin. Reaction mixtures contained the following compounds, where appropriate, in a final volume of 1.2 ml of 1 mM phosphate buffer (FH 7.4): chromatin dialyzed against phosphate buffer (0.12 mg DNA/ml), HrOr (2.8 mM), FeC& (25 PM), CuSO, (25 NM), EDTA (100 PM), NTA (100 @M), ascorbic acid (100 pM), mannitol (50 mM), DMSO (50 mM), SOD (200 u/ml). Where indicated, FeC& and CuSOl were mixed with EDTA or NTA prior to addition to the reaction mixture. Chelex-treated phosphate buffer (1 mM, pH 7.4) was used for all dilutions. Mixtures were incubated at 37°C for 1 h. After incubation, 1 nmol each of 6-azathymine and 8-azaadenine were added as internal standards to each of the chromatin samples containing 0.12 mg DNA. Samples were immediately frozen in liquid nitrogen and lyophilized. Hydrolysis with formic acid of lyophilized samples of chromatin, trimethylsilylation of hydrolysates, and analysis of trimethylsilylated hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring were performed as described previously (28).

RESULTS The isolated chromatin was analyzed by gel electrophoresis to evaluate the authenticity of its protein components. Commercially available histones were used as reference compounds. Figure 1 illustrates the electrophoretie patterns of isolated chromatin dialyzed against phosphate buffer (lane 1) and of histones (lane 2). The electrophoretic patterns in Fig 1 are quite similar to those of histones published previously (35). Figure 1 clearly illustrates that histones Hl, H3, H2B, H2A, and H4 were present in isolated chromatin. No other proteins were detected with significant amounts. The absorption spectrum

HYDROGEN

PEROXIDE-INDUCED

DNA

DAMAGE

IN

MAMMALIAN

CHROMATIN

319

pounds identified in untreated chromatin (Table IA, column 2), showing that dialysis of isolated chromatin against Chelex-treated phosphate buffer containing EDTA had removed most of the metal ions from chromatin. This observation is consistent with our previous observation that HzOz alone does not cause any base damage in DNA (10, 14).

H3 H2B H2A

H4

I FIG. 1. chromatin H3, H2B,

Sodium dodecyl sulfate-polyacrylamide dialyzed against phosphate buffer H2A, and H4 (lane 2).

gel electrophoresis (lane l), and histones

of Hl,

of isolated chromatin (not shown here) was similar to that published previously (28). Trimethylsilylated hydrolysates of chromatin were analyzed by GC/MS-SIM. The use of this technique for identification and quantitation of free radical-induced base products in DNA and in chromatin has been described previously (28, 32, 33, 36). Figure 2 illustrates a representative selected-ion chromatogram obtained during the GC/MS-SIM analysis of a trimethylsilylated hydrolysate of chromatin exposed to HzOz in the presence of Fe3+-NTA. Peak identification is given in the figure legend. The DNA base products identified in chromatin and their yields measured under various conditions are listed in Tables IA, IB, IIA, and IIB. With the exception of &OHMeUra, all the compounds identified in treated samples were also found in untreated chromatin (Table IA, column 1). Incubation of chromatin with H202 alone did not produce any increase in the amount of the com-

The effect of ferric ions. Treatment of chromatin with Fe3+ ions alone did not increase the amounts of base modification (data not shown). HeOz with Fe3+ produced a slight increase in the amounts of all the compounds over the background levels, and also caused formation of traces of 5OHMeUra (Table IA, column 4). When ascorbic acid was present in the reaction mixture containing H202/Fe3+, a substantial increase in product yields was observed with a particularly high proportional increase in the yields of formamidopyrimidines over the background levels (Table IA, column 5). No increase in product yields was observed except for the slight increase in the yields of formamidopyrimidines when ascorbic acid and H202 without Fe3+ ions were present in the reaction mixture (Table IA, column 3), again indicating successful removal of contaminating metal ions from chromatin. Addition of SOD to the reaction mixture containing H202/Fe3’/ascorbic acid did not affect the product yields (Table IA, column 6). Mannitol and DMSO at high concentrations (50 mM) provided significant inhibition of product formation; however, the yields were still much higher than background levels (Table IA, columns 7 and 8). Treatment of chromatin with H202/Fe3’-EDTA produced higher yields of the products than that with H,O,/ Fe3’ (compare Table IA, column 4 and Table IB, column 9). Addition of ascorbic acid to this system caused an increase in the yields of most products with the highest proportional increase being in the yields of formamidopyrimidines (Table IB, column 10). H202/Fe3+-NTA produced more base damage than H,02/Fe3+-EDTA (Table IB, column ll), in agreement with our previous observations upon isolated DNA (14). Addition of ascorbic acid to the H,02/Fe3+-NTA system increased the yields of all the products (Table IB, column 12). The presence of SOD in this system caused a marked inhibition of product formation in contrast to the H,0,/Fe3+/ascorbic acid/SOD system (compare Table IA, column 6 and Table IB, column 13). The presence of mannitol and DMSO decreased the yields of the products to almost background levels. This is in contrast to the observation with HZOz/Fe3+/ ascorbic acid/mannitol and with Hz0,/Fe3+/ascorbic acid/ DMSO (compare Table IA, columns 7 and 8, and Table IB, columns 14 and 15). The effect of cupric ions. Treatment of chromatin with CL?+ ions alone caused an increase in the amounts of all the compounds with a substantial increase in the amounts of B-hydroxypurines (Table IIA, column 2). The chelation of cu2+ with NTA decreased the product formation;

320

DIZDAROGLU

ET

AL.

20000-

14

1800016000-

6

14000-

FIG. 2. Ion-current profiles obtained during the GC/MS-SIM analysis of a trimethylsilylated hydrolysate of mammalian chromatin treated with H202 in the presence of Fe3+-NTA. For experimental details see Materials and Methods. The column was programmed from 150 to 260°C at a rate of 8”C/min after 2 min at 150°C. The amount of an aliquot of DNA in chromatin injected onto the column was approximately 0.4 pg. Peaks (ion monitored): 1,6-azathymine (m/z 271) (internal standard); 2, 5-OH-5-MeHyd (m/z 331); 3,5-OH-Hyd (m/z 317); 4, 5-OH-Ura (m/z 329); 5, 5-OHMeUra (m/z 358); 6, B-OH-Q& (m/t 343); 7, &-Thy glycol (m/z 259); 8, 5,6-dihydroxyuracil (m/z 417); 9, truns-Thy glycol (m/z 259); 10, 8-azaadenine (m/z 265) (internal standard); 11, FapyAde (m/z 354); 12, 8-OH-Ade (m/z 352); 13, FapyGua (m/z 442); 14, 8-OH-Gua (m/z 440) (all compounds as their trimethylsilyl derivatives). [It should be noted that 5-OH-Ura (peak 4) and 5-OH-Cyt (peak 6) arise by acidinduced modification of Cyt glycol. Similarly, 5,6-dihydroxyuracil (peak 8) is produced by acid-induced deamination of 5,6-diOH-Cyt. 5-OH-5MeHyd (peak 2) and 5.OH-Hyd (peak 3) are believed to result from acid-induced modification of 5-methyl-5hydroxybarbituric acid and 5,6diOH-Cyt, respectively.]

background levels of the amounts of most products were observed (Table IIA, column 3). Treatment of chromatin with Hz02/Cu2+ caused extensive damage to the DNA bases, much more than the treatment with H202/Fe3+ (compare Table IA, column 4 and Table IIA, column 4). Addition of ascorbic acid to the reaction mixture caused

approximately a twofold increase in the yields of most products (Table IIA, column 5). The yields of FapyAde and FapyGua were increased by about threefold. The presence of SOD, mannitol, and DMSO in the reaction mixtures did not inhibit product formation (Table IIA, columns 6, 7, and 8).

TABLE

IA

Yields (nmol/mg of DNA”) of DNA BaseProducts Formed in Chromatin by Treatment with the HzOz/Fe3+System Treatment lb

Product 5-OH-5-MeHyd 5-OH-Hyd cyt glycol 5-OHMeUra Thy glycol 5,6-diOH-Cyt FapyAde 8-OH-Ade FapyGua 8-OH-Gua

2

0.11 -c 0.02 0.23 0.55

+_ 0.014 f 0.06

n.d.c

0.10 0.24 0.56

+ 0.005 -c 0.014 ?I 0.06

0.11 f 0.001

n.d. 0.12 f 0.03 0.09 t 0.003

0.15 0.28 0.05 0.57

0.15 0.28 0.06 0.53

0.08

n All values represent b 1, chr; 2, chr/H,O*; chr/H,OJFea+/asc/DMSO. ’ Not detected.

-c 0.015 f rt_ ?I f

0.006 0.018 0.017 0.013

f 0.02

+ 0.01 f 0.02 + 0.04

3

4

0.11 + 0.016 0.22 0.54

5~ 0.018 I!z 0.02

n.d. 0.07 0.07 0.30 0.33 0.20 0.83

the mean * standard deviation 3, chr/H,O,/asc; 4, chr/HzO,/Fe

-c 0.007 + 0.005 f

0.05

f 0.01 k

0.05

+ 0.06

0.19 + 0.006 0.29 + 0.027 1.30 * 0.155 0.03 + 0.01 0.16 + 0.025 0.30 k 0.016 0.30 f 0.04 0.91 + 0.057 0.21 f 0.06 1.73 +- 0.38

5

f 0.015 0.39 f 0.02 4.38 + 0.10 0.09 f 0.013 0.79 2 0.07 0.84 2 0.05 2.18 i 0.084 2.57 3~ 0.06 1.99 * 0.33 12.0 + 0.77 0.60

6 0.58 0.52 4.48 0.13 0.85 0.62 2.58 2.64

7

f 0.05 + 0.05 f 0.18 + 0.01

f 0.146 + 0.085 f 0.37

k 0.19 1.95 * 0.20 12.4 f 0.18

from triplicate measurements. 3+., 5 , chr/H,0,/Fe3’/asc; 6, chr/HzOz/Fe3+/asc/SOD;

0.25 0.32 1.60 0.03 0.28 0.28 0.84

8

3~ 0.03 +_ 0.03 + 0.15 -c 0.004 f

0.03

+ 0.02 f

0.07

0.98 + 0.10 1.02 f 0.11 5.60

2~ 0.60

7, chr/H202/Fe3+/asc/mannitol;

0.28 0.30

f 0.07 * 0.03

1.79 + 0.29 + 0.01

0.04 0.23 0.24 0.79 0.94 0.96 5.89

+ 0.06 2~ 0.013 + 0.097 f 0.22 zk 0.25 + 0.37

8,

HYDROGEN

PEROXIDE-INDUCED

DNA

DAMAGE

TABLE

Yields (nmol/mg

of DNA’)

of DNA Base Products

Formed

IN

MAMMALIAN

321

CHROMATIN

IB

in Chromatin

by Treatment

with the H,O,/Chelated

Fe3+ System

Treatment Product 5-OH-B-MeHyd 5-OH-Hyd cyt glycol 5-OHMeUra Thy glycol 5,6-diOH-Cyt FapyAde S-OH-Ade FapyGua 8-OH-Gua

96 0.29 0.30 2.77 0.07 0.17 0.45 0.57 2.06 0.38 3.40

10

f 0.025 f 0.01 _+ 0.26 f 0.009 zk 0.048 + 0.06 + 0.09 + 0.23 rt 0.006 f 0.34

0.48 0.35 3.45 0.07 0.59 0.71 1.66 1.06 2.03 4.95

f k f f + f k + f +

11 0.006 0.017 0.21 0.004 0.09 0.045 0.013 0.015 0.07 0.11

0.56 0.41 5.54 0.10 0.82 0.85 1.77 2.65 0.78 6.60

12

+ 0.05 f 0.026 Ifr 1.14 _+ 0.014 f 0.18 f 0.08 + 0.09 f 0.25 f 0.15 + 0.34

0.84 0.47 6.68 0.15 1.78 1.23 2.68 3.34 1.75 9.57

* f f f + + f f k +

0 All values represent the mean f standard deviation from triplicate measurements. * 9, chr/H,0Z/Fe3+-EDTA; 10, chr/HeO.JFes+-EDTA/asc; 11, chr/H,O,/Fee+-NTA; SOD; 14, chr/H,02/Fe3+-NTA/ asc / mannitol; 15, chr/Hz0,/Fe3+-NTA/asc/DMSO.

TABLE

(nmol/mg

of DNA”)

of DNA Base Products

0.005 0.017 0.50 0.026 0.08 0.015 0.40 0.08 0.22 0.61

0.44 0.52 4.31 0.10 0.62 0.37 0.94 1.54 1.23 5.03

+ + 2 + k * f + k f

14 0.035 0.12 0.68 0.02 0.03 0.10 0.138 0.086 0.04 0.15

12, chr/H,0z/Fe3+-NTA/asc;

0.15 0.36 0.85 0.06 0.15 0.11 0.25 0.40 0.20 0.88

15

+ 0.001 f 0.03 f 0.07 k 0.005 AZ 0.01 f 0.005 k 0.02 * 0.05 2 0.025 f 0.09

0.16 0.31 0.74 0.05 0.13 0.07 0.26 0.36 0.18 0.75

+ k f f + k 5 -t + +

0.015 0.017 0.038 0.01 0.014 0.016 0.06 0.065 0.047 0.07

13, chr/H,0,/Fe3+-NTA/asc/

yields were still lower than those in chromatin treated with H202/Cu2+/ascorbic acid with the exception of the yields of FapyAde and FapyGua (compare Table IIA, column 5 and Table IIB, column 12). SOD provided no product inhibition, and even increased the yields of most products. The presence of mannitol and DMSO caused only a slight decrease in the yields of the products. Comparison with calf thymus DNA. Treatment of calf thymus DNA under the same conditions and using the same amount of DNA as in chromatin produced higher yields of products than those in chromatin. Table III shows the yields of the products in calf thymus DNA under some selected conditions. As in chromatin, H202/Fe3+NTA/ascorbic acid caused more damage in calf thymus

Chelation of CL?+ with EDTA inhibited product formation (Table IIB, column 9). The addition of ascorbic acid to H20&u2+-EDTA slightly increased the yields of formamidopyrimidines and 8-hydroxypurines. When NTA was used instead of EDTA for chelation of Cu2+, only the yields of Cyt glycol, 8-OH-Ade, and 8-OH-Gua were increased. The damage done by H202/Cu2+-NTA was much less than that by HzOz/Cu2+ (compare Table IIA, column 4 and Table IIB, column ll), in contrast to the results obtained with H202/Fe3+-NTA and Hz02/Fe3+. However, the addition of ascorbic acid to the reaction mixture caused extensive damage with the highest proportional increase in the yields of FapyGua and &OHGua (approximately lo-fold) (Table IIB, lane 12). The

Yields

13

IIA

Formed

in Chromatin

by Treatment

with the H202/Cu2+

System

Treatment Product 5-OH-5-MeHyd 5-OH-Hyd cyt glycol 5-OHMeUra Thy glycol 5,6-diOH-Cyt FapyAde 8-OH-Ade FapyGua 8-OH-Gua

lb 0.11 * 0.02 0.23 f 0.014 0.55 -c 0.06 n.d.’ 0.08 f 0.015 0.11 f 0.001 0.15 + 0.006 0.28 + 0.018 0.05 k 0.017 0.57 f 0.013

2 0.15 0.30 1.92 0.07 0.22 0.05 0.21 3.03 0.14 3.54

f tf + 2 f + k * 2

3 0.014 0.017 0.02 0.01 0.05 0.005 0.01 0.19 0.03 0.27

0.10 k 0.002 0.30 2 0.016 0.56 f 0.01 n.d. 0.07 AZ 0.007 0.03 + 0.005 0.15 f 0.017 0.70 + 0.06 0.07 f 0.002 0.76 f 0.14

4 0.36 0.38 5.59 0.10 0.55 0.15 0.59 8.48 0.35 15.1

f 0.06 + 0.026 _+ 0.54 + 0.006 k 0.19 ? 0.018 + 0.13 A 0.97 5 0.10 +- 1.12

5 0.52 0.56 10.7 0.21 0.95 0.29 1.51 15.4 1.11 34.0

f 2 f f * f f + +IL

6 0.013 0.136 0.75 0.011 0.11 0.05 0.024 1.21 0.17 1.85

0.56 0.60 10.8 0.24 1.00 0.25 1.71 17.6 1.17 35.1

a All values represent the mean f standard deviation from triplicate measurements. * 1, chr; 2, chr/Cu *+; 3, chr/Cu*+-NTA; 4, chr/H202/Cu2+; 5, chr/Hz0,/Cu2+/asc; 6, chr/HzOz/CuZ+/asc/SOD; 8, chr/H202/Cu2+/asc/DMS0. ’ Not detected.

t 2 f + tf + rt f *

7 0.02 0.012 0.30 0.02 0.15 0.05 0.12 0.7 0.06 1.99

0.44 0.38 7.48 0.23 1.07 0.29 1.42 13.1 0.45 34.3

f 2 + f k * + * k +

8 0.034 0.023 0.214 0.02 0.16 0.04 0.155 1.10 0.19 3.15

0.36 0.44 9.61 0.20 1.05 0.25 1.52 13.8 0.84 36.0

7, chr/H,O,/CW/asc/mannitol;

+ k + + zk k + k k f

0.04 0.027 0.33 0.023 0.05 0.02 0.26 1.12 0.19 4.07

322

DIZDAROGLU TABLE

Yields (nmol/mg

of DNA”)

of DNA Base Products

Formed

ET AL. IIB

in Chromatin

by Treatment

with the HrOJChelated

Cu2+ System

Treatment Product 5OH-5-MeHyd 5-OH-Hyd cyt glycol 5-OHMeUra Thy glycol 5,6-diOH-Cyt FapyAde 8OH-Ade FapyGua 8-OH-Gua

10

gb

0.15 zk 0.02 0.27 + 0.01 0.60

0.14 + 0.02 0.28 f 0.01 0.63 + 0.10 0.02 zk 0.005 0.06 2~ 0.014 0.05 f 0.01 0.49 + 0.035 0.44 2 0.05 0.37 f 0.05 0.72 & 0.02

f 0.002

n.d. 0.08 0.06

IL 0.013

0.28 0.14 0.52

+ 0.025 * 0.03 k 0.09

k 0.01 0.19 * 0.014

11 0.17 + 0.30 f 2.53 + 0.05 f 0.11 + 0.05 f 0.28 2 1.75 f 0.17 + 2.18 +

0.01 0.01 0.37 0.017

0.028 0.01 0.038 0.41 0.028 0.28

12

13

14

15

-c 0.02 f 0.038 2 0.25 ix 0.02 0.59 * 0.04 0.22 2 0.04 1.80 T 0.11 3.90 f 0.06 1.46 f 0.11 23.7 f 0.68

xi 0.015 2 0.008 7.93 3~0.24 0.15 IL 0.018 0.58 5~0.07 0.21 f 0.02 2.25 510.13 4.75 2 0.16 1.95 zt 0.18 28.1 + 0.80

f 0.014 + 0.02 f 0.07 0.11 + 0.01 0.52 t 0.03 0.16 f. 0.03 1.49 + 0.08 3.42 xk0.12 0.59 + 0.18 17.3 It 1.38

0.22 Ik 0.01 0.29 2 0.01 4.42 2 0.29 0.12 f 0.01 0.25 f 0.047 0.06 + 0.01 1.21 k 0.06 2.98 +- 0.03 0.86 * 0.09 19.8 + 0.91

0.40 0.34 7.63 0.13

o All values represent the mean + standard deviation from triplicate measurements. ’ 9, chr/H10z/Cu2+-EDTA, 10, chr/H,Or/C u’+-EDTA/asc; 11, chr/H20r/Cu2+-NTA, ax/SOD; 14, chr/HI0,/Cu2+-NTA/asc/mannitol; 15, chr/H,O,/Cu*+-NTA/asc/DMSO.

DNA than H202/Fe3+/ascorbic acid, and the damage by HzOz/Cu2+/ascorbic acid was higher than that by H202/ Cu2+-NTA/ascorbic acid. DISCUSSION

The results in the present study show that modified bases identified previously in DNA are also formed in mammalian chromatin upon treatment with H202 in the presence of chelated and unchelated Fe3+ or Cu2+. The pattern of base modification is consistent with the idea that ‘OH is responsible for the formation of the DNA baseproducts identified in chromatin in the present work. Inhibition of product formation in the presence of chelated Fe3+ and Cu2+ by typical scavengers of ‘OH supports this idea. When unchelated Fe3+ or Cu2+ were used in the re-

Yields (nmol/mg

of DNAa) of Base Products

0.43 0.31 4.03

12, chr/H,O,/Cu*+-NTA/asc;

13, chr/HrOJCu*+-NTA/

action mixtures, however, scavengers provided partial inhibition in the presence of Fe3+ or no inhibition in the presence of Cu 2+. This might be due to binding of the unchelated ions to chromatin and formation of ‘OH at the binding sites in close proximity of DNA, so that the ‘OH reacts with DNA basesin chromatin rather than with scavengers (12, 37). There were major differences between the effects of the chelated and the unchelated forms of the same ion, and between the effects of Fe3+and Cu2+on product formation. H202 with EDTA- or NTA-chelated Fe3+ caused more DNA base damage than H202 with unchelated Fe3+. By contrast, H202 with EDTA- or NTA-chelated Cu2+ produced lower yields of products than H202 with unchelated Cu2+. Essentially no increase in the product yields over the background levels was observed when H202/Cu2+-

TABLE III in Calf Thymus

Formed

0.41 0.47

DNA by Treatment

with H202 under Various

Conditions

Treatment Product 5-OH-5-MeHyd 5-OH-Hyd cyt glycol 5-OHMeUra Thy glycol 5,6-diOH-Cyt FapyAde 8-OH-Ade FapyGua 8-OH-Gua

lb

2

0.22 0.13 0.77 0.06 0.35 0.09

f 0.014 + + + -c

0.012 0.015 0.016 0.025

0.19 0.89 0.17 1.02

+ f + f

0.05 0.16 0.01 0.10

iz 0.01

2.43

f 0.12

1.01 + 0.10 14.6 + 1.13 0.25 2.60 3.71 8.63 4.55 1.84

f 0.04 f 0.28 + 0.50

3~ 1.39 f 0.74 AZ 0.60

39.1 f 4.5

3

4

AZ0.67 + 0.08 + 0.9 + 0.06 f 4.1 +_ 1.6 + 4.0 f 0.28 1.69 -c 0.18 15.8 f 2.2

5~0.11 f 0.30 + 0.7 f 0.05 f 0.65 + 0.014 1.01 f 0.15 30.5 * 2.9 0.53 Ii 0.075 96.0 f 2.3

8.64 3.05 46.3 0.75 25.3 24.2 22.6 8.53

’ All values represent the mean f standard deviation from triplicate measurements. ’ 1, DNA; 2, DNA/H202Fe3+/asc; 3, DNA/H,O,/F e3+-NTA/asc; 4, DNA/H202/Cu2+/asc;

1.46 1.78 24.1 0.41 6.32 0.78

5, DNA/H202/Cu2+-NTA/asc.

5

+ 0.18 +- 0.16 f 1.3 f 0.015 k 0.34 + 0.015 f 0.06 + 1.6 0.94 k 0.05 40.7 f 3.5 1.58 0.85 11.5 0.15 4.03 0.85 1.57 12.5

HYDROGEN

PEROXIDE-INDUCED

DNA

EDTA was used. Chelation of both ions with NTA caused more DNA damage than that with EDTA, in agreement with previous results obtained with calf thymus DNA (14, 17). H202 with chelated Fe3+ caused more damage than Hz02 with chelated Cu2+. By contrast, damage by H202 with unchelated Cu2+ was much more extensive than that by H202 with unchelated Fe3+. When unchelated metal ions are added to a reaction mixture, they can bind to various components of the reaction mixture and lead to “site-specific” generation of reactive species such as ‘OH (37). This type of generation of ‘OH is more likely to occur in uiuo than is production of ‘OH in “free solution” that has to migrate to reach DNA. The addition of ascorbic acid to reaction mixtures generally caused an increase in the yields of the products. Ascorbic acid is an antioxidant, but it can also act as a prooxidant if metal ions are present (38). The addition of ascorbic acid to H202/Fe3+ substantially increased the yields of the products. However, the increases in product yields were not as much when chelated Fe3+ was used instead of unchelated Fe3+. Similarly, the H202/Cu2+/ ascorbic acid system caused more extensive DNA damage than the H,OJchelated Cu2+/ascorbic acid system. The presence of NTA in this system was much more effective than that of EDTA in product formation. Taken together, the results indicate that ascorbic acid increases the DNA damage by H202 in the presence of chelated or unchelated Fe3+ and Cu2+, most likely by reducing them to Fe2+ and Cu+. The highest increase occurs when Fe3+ and Cu2+ are not chelated, which may allow them to bind to the DNA in chromatin. Ferric and cupric ions, their chelation, and the presence of ascorbic acid had a pronounced effect on the ratio of the yields of 8-hydroxypurines to those of formamidopyrimidines (i.e., 8-OH-Gua/FapyGua and 8-OH-Ade/ FapyAde). 8-Hydroxypurines and formamidopyrimidines result from addition of ‘OH to the C-8 of purines followed by respective one-electron oxidation and reduction of the C-8 OH-adduct radicals of purines (39). The presence of Cu2+ may favor the oxidation of the C-8 OH-adduct radicals of purines as the following ratios suggest: 8-OHGua/FapyGua = 43; 8-OH-Ade/FapyAde = 14 (from Table IIA). These ratios were 8 and 3 (from Table IA), respectively, when Fe3+ was used instead of Cu2+. Chelation of Cu2+ markedly decreased these ratios, indicating a possible change in the reduction potential of this ion by chelation. By contrast, these ratios were not affected by chelation of Fe3+, except for the ratio 8-OH-Ade/FapyAde in the case of Fe”+-NTA. Addition of ascorbic acid to reaction mixtures generally decreased the ratios 8-OH-Gus/ FapyGua and 8-OH-Ade/FapyAde. In fact, the yield of FapyAde was higher than that of 8-OH-Ade with H202/ Fe3+-EDTA/ascorbic acid. These results suggest that ascorbic acid increases the reduction of the C-8 OH-adduct radicals of purines, in addition to its role as a reducing agent for Fe3+ and Cu2+.

DAMAGE

IN

MAMMALIAN

CHROMATIN

323

SOD provided some inhibition of product formation when the H202/Fe3+-NTA/ascorbic acid system was used, implicating the involvement of 0; in the generation of ‘OH, in agreement with a previous observation (14). In the other cases, however, no product inhibition by SOD was observed. In fact, addition of SOD to the reaction mixture slightly increased the yields of the products in the case of Cu2+-mediated DNA damage. It seems that 0; is not required for Cu2+- or unchelated Fe3+-mediated generation of ‘OH. Except for &OHMeUra, all the DNA products produced in chromatin upon treatment with H202 in the presence of ferric or cupric ions were also found to be present in untreated chromatin. The amounts of these compounds were generally lower than those found in calf thymus DNA. The presence of these compounds in DNA from various sources has also been reported previously (10,14, 40, 41) and may represent intrinsic damage to DNA due to free radical formation in duo. On the other hand, the procedure for isolation of chromatin may also contribute in part to the formation of the products. However, it should be noted that this procedure is milder than that used for isolation of DNA in the aforementioned papers. Finally, the acidic hydrolysis, which is an essential part of the methodology used in the present work, might also contribute to the background levels of modified DNA bases in chromatin. The yields of the modified DNA bases were substantially lower than the yields of the same modified bases found in isolated calf thymus DNA treated with H202 in the presence of ferric or cupric ions under the same experimental conditions. This may be because of the ability of histones in chromatin to scavenge ‘OH, so protecting the DNA. Histones might also restrict the binding of metal ions to DNA. They could additionally affect formation of the products measured. For example, they might affect the oxidation and reduction of OH-adduct radicals of pyrimidines and purines. Furthermore, the DNA bases might participate in the formation of DNA-protein cross-links in the chromatin (26, 30, 42), resulting in the decreased yields of the products. For the reasons outlined above, the present study suggests that mammalian chromatin should be considered as a different system from DNA alone when studying the effect of free radical-producing systems. Furthermore, the data indicate that histones in chromatin may not be able to prevent metal ion-dependent free radical damage to DNA in duo. In conclusion, typical ‘OH-induced products of DNA bases were identified and quantitated in mammalian chromatin treated with H202 in aqueous suspension in the presence of cupric and ferric ions. The GC/MS-SIM technique permits the unequivocal identification and quantitation of the DNA base products in chromatin without the necessity to isolate the DNA from the chromatin. The types of products and inhibition of their formation by typical scavengers of ‘OH strongly suggest the

324

DIZDAROGLU

involvement of ‘OH in product formation. Substantial differences exist between the actions of ferric and cupric ions, and between the actions of the chelated and unchelated ions. The presence of ascorbic acid generally increases the yields of the products, and favors the reduction of C-8 OH-adduct radicals of purines. The yields of the products are lower than those observed with DNA alone, indicating the protecting effect of histones in chromatin. ACKNOWLEDGMENTS This work was supported in part by the Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy, Washington, DC. G.R. acknowledges support from the National Science Foundation (EET-8808775). We thank B. C. Chao for assistance in growing the cells. B.H. is grateful to the Medical Research Council and the Arthritis and Rheumatism Council, U.K. for research support.

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