Oxidative damage to DNA in mammalian chromatin

Mutation Research, 275 (1992) 331-342 331 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8734/92/$05.00

MUTAGI 0260

Oxidative damage to DNA in mammalian chromatin

Miral D i z d a r o g l u Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

(Received 21 April 1992) (Revision received 16 June 1992)

(Accepted 16 June 1992)

Keywcrds: DNA damage; Ageing; Hydroxyl radical; Modified DNA bases; DNA-protein cross-links; Oxygen effect

Summary

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivo by a variety of free radical-producing systems. Methodologies incorporat- ing the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H 20., and in chromatin of organs of animals treated with carcinogenic metal salts.

Correspondence: Dr. M. Dizdaroglu, National Institute of Standards and Technology, Bldg. 222/A353, Gaithersburg, MD 20899, USA.

Abbreriations: 0 ~ , superoxide radical; "OH, hydroxyl radical; e~, hydrated electron; 5,6-diHThy, 5,6-dihydrothymine; 5- OH-5-MeHyd, 5-hydroxy-5-mcthylhydantoin; 5-OH-Hyd, 5- hydroxyhydantoin; 5-OH-Ura, 5-hydroxyuracil; 5-OH-6-HThy, 5.hydroxy-6.hydrothymine; 5-OH-6-H(':,t, 5-hydroxy-6.hydrocytosine; 5-OHMeUra, 5.(hydroxymethyl)uracil; 5-OH-Cyt, 5- hydroxycytosine; Thy glycol, thymine glycol; Cyt glycol, cyto. sine glycol; 5,6-diOH-Ura, 5,6-dihydroxyuracil; 5,6-diOH-Cyt, 5,6.dihydroxycytosine; FapyAde, 4,6.diamino-5-formamidopyrimidine; 8-OH-Ade, 8.hydroxyadenine; 2-OH-Ade, 2-hydroxyadenine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8-OH-Gua, 8.hydroxyguanine; GC/MS-SIM, gas chromatography/mass spectrometry with selected-ion monitoring; DMSO, dimethyl sulfoxide; SOD, superoxide dis- mutase; SD, standard deviation.

Oxidative damage caused by free radicals in vivo has been implicated to play an important role in the occurrence of biological processes such as mutagenesis, carcinogenesis and aging (for a review see Halliweli and Gutteridge, 1989). Free radicals may be produced in vivo by endoge- nous and exogenous sources and cause damage to biological molecules including DNA by a variety of mechanisms (for a review see Halliwell and Aruoma, 1991). Thus free radicals may be muta- genie and carcinogenic (for a review see Breimer, 1990). Oxygen-derived species such as superoxide radical (Of) and H20 e are generated in all aerobic cells (Fridovich, 1986; Halliwell and Gut- teridge, 1989). Neither 0 2 nor HeO 2 under physiological conditions appear to cause DNA

332

damage (Lesko et al., 1980; Aruoma et al., 1989; Blakely et al., 1990). Thus, the toxicity of these species has been attributed to the highly reactive hydroxyl radical ('OH), which can be formed by metal ion-catalyzed reactions of Of and H20 2 (Halliwell and Gutteridge, 1989). Ionizing radiation can also produce "OH and other radical species (i.e., hydrated electron (eaq) and H atom) in vivo by interaction with cellular water (for a review see yon Sonntag, 1987). In oxic cells, the contribution of free radicals to the lethal action and DNA damage by ionizing radiation amounts to = 70% (Roots and Okada, 1972, 1975; Chap- man et al., 1973). Hydroxyl radical produces a number of lesions in DNA and in nucleoprotein such as base lesions, sugar lesions, single-strand breaks, double-strand breaks, abasic sites and DNA-protein cross-links by a variety of mechanisms (for reviews see T6oule and Cadet, 1978; yon Sonntag, 1987; Oleinick et ai., 1987; Steenken, 1989). Ionizing radiation-generated e~ and H atom can also modify DNA bases (yon Sonntag, 1987; Steenken, 1989). Lesions produced in DNA in vivo are subject to cellular repair processes and

CHs ~. CHs Ht, I~_CHHs H .X' "~OH XX 'r ~-'OX

$.hydroxyo6-hydrothymine |1~oi $,6.d|l~drothymlne thymine

can be remo~,ed from DNA; however, failure of DNA repair may result in serious biological consequences (for a review see Friedberg, 1985).

The present article reviews recent results on free radical-induced DNA damage in mammalian chromatin in vitro and in vivo.

DNA damage in chromatin in vitro

We have investigated free radical-induced DNA damage in chromatin with the use of the technique of gas chromatography/mass spectrometry (GC/MS). This technique permits the identification and quantitation of a large number of pyrimidine- and purine-derived DNA bases in chromatin in the presence of proteins without the necessity of isolating DNA from chromatin. A number of DNA-protein cross-links can also be characterized and quantitated (for a review see Dizdaroglu, 1991).

DNA base damage produced by ionizing radiation Hydroxyl radical, e~ and H atom are pro-

duced when water is exposed to ionizing radia.

H 0 H ~ , ~ C H | Q H O ~ C # s Or,,,~,.,"-,H

S-(h,vdroxymethyl). $-hydroxy-S.methyl- ursdl hydantoln

xx¢ N Xt

S-hydroxy.6-hydrocytosine ~toslne |!Icol

O•,,,HN• H H H OH O

O

S-hydrny. S-hydroxy- S,6-dihydrox~, S,6.dihydro~. S.hyclroxyhy~hlntoin cytosine uracil uracil qtosine

N.H2

H

8-hydroxysdenlne

NH| N ~ N NH'C~

H~

4,6.,dlamlno-$-rormllmido., pyrimldin~

140' H HIN O14 HIN'~It.'~"NII| H

2-hydroxTadenine 8-hydroxTipumine 2,@dlemlno-4-h).drox) - s-rormamldopyrimidine

Fig, I, Structures of DNA base products,

333

tion (for a review see yon Sonntag, 1987). In the presence of oxygen, e~a and H atom react with oxygen at diffusion-controlled rates and are converted into 0 2 . When N20 is present in solution, e~q reacts with N20 at a diffusion-controlled rate to yield additional "OH. For investigation of ionizing radiation-induced DNA damage in isolated chromatin, aqueous suspensions of chromatin were saturated with argon, air, N20 and N20/O2 and then exposed to ionizing radiation (Gajewski et al., 1990). Under these conditions, the radical species produced and their yields (~mol/J) are as follows: argon: "OH (0.28), e~'q (0.27), H atom (0.057); air: "OH (0.28), 0 2 (0.33); N20: "OH (0.56), H atom (0.057); N20/O2: "OH (0.56), 02 (0.057) (for a review see yon Sonntag, 1987). Trimethyisilylated hydrolysates of chromatin samples were analyzed by GC/MS-SIM. The following modified bases were identified and their radiation yields were measured under aforemen- tioned gaseous conditions: 5,6.dihydrothymine, 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, cytosine glycol, 5-hydroxy-6-hydrothymine, 5- hydroxy-6-hydrocytosine, 5-(hydroxymethyl)uracil, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8.hydroxyadenine, 2,6.diamino-4-hydroxy-5-formamidopyrimidine and 8.hydroxyguanine (see Fig. 1 for the structures of these modified DNA bases) (Gajewski et al., 1990). AFI products yielded linear dose-yield relationships. The formation of modified bases and their yields depended on the radical environ- ment and oxygen in the aqueous system. In the presence of oxygen (air and N ,O/O, ) , the formation of 5,6-diHThy, 5-OH-6-HThy, and 5-OH- 6-HCyt was inhibited. This is because 5,6-diHThy arises from reactions of eaq and H atom with thymine (T6oule and Cadet, 1978; yon Sonntag, 1987). The formation of 5-OH-6-HThy and 5- OH-6-HCyt is inhibited due to the diffusion-controlled reaction of oxygen with their precursors, namely C5-OH-adduct radicals of thymine and cytosine (T~oule and Cadet, 1978; von Sonntag, 1987). Cyt glycol, Thy glycol and 5-OHMe-Ura, and all the products of purines were observed under all four gaseous conditions. Cyt glycol and Thy glycol were produced more in the presence of oxygen than in its absence. This may be due in part to inhibition by oxygen of the formation of

5-OH-6-hydropyrimidines, which result from OH-adduct radicals of pyrimidines as do pyrimidine glycols. The yields of both 8-OH-Ade and 8-OH-Gua were higher in the presence of oxygen than in its absence. However, oxygen did not affect the yields of formamidopyrimidines, which were even higher than those of 8-hydroxypurines in the absence of oxygen. These purine products result from addition of "OH to the C8 of purines followed by respective one-electron reduction and oxidation of the C8-OH-aduuct radicals (for a review see Steenken, 1989). The results indicate that oxygen strongly favored the oxidation of the C8-OH-adduct radicals of purines resulting in formation of 8-hydroxypurines. The formation of formamidopyrimidines in the presence of oxygen was not suppressed. This is in contrast to inhibition by oxygen of formation of pyrimidine products 5-OH-6-HThy and 5-OH-6-HCyt, which result similarly from reduction of corresponding CS-OH-adduct radicals of pyrimidines.

DNA base damage produced by hydrogen peroxide and metal ions

Hydrogen peroxide is ubiquitous in biological systems and is formed in all aerobic cells as a result of normal cellular metabolism (for a review see Halliwell and Gutteridge, 1989). Hydrogen peroxide is also formed by interaction of ionizing radiation with water (for a review see yon Sonn- tag, 1987). Unlike 'OH, which has a short diffusion distance in cells due to its high reactivity toward organic molecules and thus reacts at or close to its site of formation, H20 2 can diffuse long distances in cells to reach DNA and can also cross biological membranes. Most of the "OH generated in vivo, except during exposure to ionizing radiation, results from transition metal ion- catalyzed conversion of H 202 (Halliwell ad Gut- teridge, 1989).

Recently, we have investigated the effect of H202 in the presence of various transition metal ions such as Fe(IIl), Cu(II), Co(ll) or Ni(II) on isolated chromatin. The following modified bases were identified and their yields were measured: 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, cytosine glycol, 5-(hydroxymethyl)uracii, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyade-

334

nine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5- formamidopyrimidine and 8-hydroxyguanine (Diz- daroglu et al., 1991b; Nackerdien et al., 1991a). Hydrogen peroxide in the presence of Cu(II) caused more DNA damage than in the presence of Fe(Ill). Chelation of Cu(II) with EDTA or nitrilotriacetic acid (NTA) caused a marked inhibition in product formation. By contrast, Fe(III)- EDTA and Fe(III)-NTA produced higher product yields than unchelated Fe(III). Product formation was significantly inhibited by typical scavengers of "OH. This was more prominent in the presence of chelated metal ions than unchelated metal ions, indicating a po~ibl,: site-specific formation of "OH when H20 2 reacts with chromatin-bound metal ions. Addition of ascorbic acid to reaction mixtures generally increased the product yields.

Product formation by Co(II)/H20 2 and Ni(II)/H20 2 was partially inhibited by scavengers of 'OH indicating the involvement of "OH in product formation. In both cases, addition of ascorbic acid to reaction mixtures did not affect the product yields. Co(II)/H20 2 caused more DNA damage than Ni(II)/H20 2. Chelation of Ni(il) and Co(ll) almost completely inhibited product formation.

Modified DNA bases identified in chromatin treated with H202 in the presence of Fe(Ill), Cu(ll), Co(ll) or Ni(II) were typical products of reactions of 'OH with DNA bases. Inhibition of product formation by typical scavengers of 'OH suggests the involvement of 'OH in product formation. Partial inhibition of product formation in the presence of 'OH scavengers may be due to binding of the unchelated ions to chromatin and formation of 'OH in a site-specific manner at the binding sites in close proximity of DNA, so that the 'OH reacts with DNA bases in chromatin rather than with scavengers (Samuni et al., 1983; Goldstein and Czapski, 1986; Ward et al., 1985). There were marked quantitative differences between the effects of the chelated and unchelated forms of the same ion, and between the effects of individual ions on product formation. The results suggest that ascorbic acid increases the reduction of the C8-OH-adduct radicals of purines, in addition to its role as a reducing agent for Fe(III) and Cu(ll). Ni(ll) alone caused significant rises in the

background amounts of modified DNA bases in chromatin. This may be due to the ability of complexes of Ni(lI) with certain peptide sequences in chromatin to generate free radicals in the presence of oxygen (Kasprzak and Bare, 1989). Failure of catalase to inhibit the damage by Ni(I1) alone indicated that HeO 2 may not be required for such reactions. On the other hand, H202/ Co(II) caused more damage in chromatin than H eO2/Ni(II). In the latter case, a substantial increase in Ni(II) concentration and in treatment time was necessary to produce significantly higher amounts than the background amounts of modified bases in chromatin. Addition of ascorbic acid had little effect on product yields. By contrast, ascorbic acid greatly stimulates DNA base modification produced by HeO2/Fe(III) or H202/ / Cu(II). Inhibition of product formation by chelation of Co(lI) and Ni(II) with EDTA is analogous to the results obtained with Cu(II), but in contrast to those obtained with Fe(III) under similar reaction conditions.

DNA base damage produced by doxorubicin The benzanthroquinone drug doxorubicin is

used in the treatment of several types of human cancer (Lenaz and Page, 1977; Young et al., 1981). It is cytotoxic and mutagenic in bacterial and mammalian cells (Vig, 1977; Au et al., 1981). Doxorubicin can undergo NADH dehydrogenase- catalyzed one-electron reduction to a semi- quinone, which reduces molecular oxygen to give Oi" and is converted to the quinone (Goodman and Hochstein, 1977; Doroshow, 1983). There is evidence that this type of reaction occurs in mammalian nuclei (Bachur et ai., 1982; Mimnaugh et al., 1985). The dismutation of Oi" leads to generation of H20 2 and to metal ion-dependent generation of "OH (Halliwell and Gutteridge, 1989). It is possible that the mutagenicity of doxorubicin may result in part from its NADH dehydrogenase-catalyzed reduction in close proximity of DNA, leading to DNA damage. For this reason, we have investigated DNA base modifications generated in isolated chromatin by doxorubicin (Akman et al., 1992). Typical 'OH-induced products of DNA bases were identified and their yields were measured by GC/MS-SIM. Treat- ment of chromatin with Cu(II)/NADH, Cu(II)/

335

doxorubicin, Cu( l l ) /NADH/doxorubic in or NADH/NADH dehydrogenase/doxorubicin did not enhance the amounts of modified bases above control levels. When Cu(II), NADH, NADH dehydrogenase and doxorubicin were all present in the reaction mixture, a marked increase in the amounts of modified bases was observed. In agreement with previous results (see above), chelation of Cu(II) with EDTA inhibited formation of modified bases. Typical scavengers of "OH provided no inhibition. Treatment of chromatin with Fe(II I ) /NADH/NADH dehydrogenase/ doxorubicin resulted in formation of the same modified bases. By contrast to Cu(ll), EDTA did not inhibit Fe(III)-mediated product formation. Again, "OH scavengers had no effect on product yields.

The data demonstrated that biologically rele- vant concentrations of doxorubiein generate modified bases in chromatin DNA similar to those caused by well-known "OH-generating systems. The mechanism of DNA damage depended on the presence of the flavoenzyme and a transition metal ion. The data suggested that base modifications mainly occur by site-specific generation of "OH. There were marked differences between the effects of Cu(ll) and Fe(lll). Amounts of base modifications were greater in the presence of Cu(ll) than in the presence of Fe(lll). Further- more, Cu(ll) was associated with a preference for cytosine and guanine modification in agreement with previous observations (Sagripanti and grae- mer, 1989; Yamamoto and Kawanishi, 1989; Diz- daroglu et al., 1991b). Base modifications observed may play a role in the mutagenicity of doxorubicin in vivo.

DNA-protein cross-links DNA-protein cross-links are a major type of

DNA damage that occurs in cells by exposure to various DNA-damaging agents (for a review see Oleinick et al., 1987). There is evidence that chemical bonds involved in DNA-protein cross- linking are of a covalent nature (Olinski et al., 1981; Mee and Adelstein, 1981; Lesko et al., 1982; Cress and Bowden, 1983; Oleinick et al., 1987). Hydroxyl radical appears to be involved in formation of DNA-protein cross-links induced by ionizing radiation or H2Oz/metal ions (Mee and

Adelstein, 1981; Lesko et al., 1982; Oleinick et al., 1987). However, little is known about the chemical nature of DNA-protcin cross-links. Re- cently, attempts have been made to determine the chemical nature of "OH-induced DNA-protein cross-links in mammalian chromatin (Gajew- ski et al., 1988; Margolis et al., 1988; Dizdaroglu et al., 1989; Dizdaroglu and Gajewski, 1989; Gajewski and Dizdaroglu, 1990; Nackerdien et al., 1991b). Since there were no authentic materi- als available for this type of molecules, model systems consisting of a y-irradiated mixture of a DNA base and an amino acid were studied first. Using this approach, the gas chromatographic and mass spectrometric properties of possible DNA base-amino acid cross-links were obtained. This information including retention times and typical fragment ions was then used to identify various DNA-protein cross-links by OC/MS-SIM in trimethylsilylated HCl-hydrolysates of chromatin. A number of DNA-protein cross-links were identified and quantitated in chromatin samples, which were y-irradiated in N20-saturated aqueous suspension. These involved the DNA bases thymine and cytosine, and the amino acids glycine, alanine, valine, leucine, isoleucine, threonine, lysine and tyrosine. In chromatin samples y-irradiated under oxic conditions (with N20/O,. bub- bling), oxygen inhibited the formation of DNA- protein cross-links except for that of the Thy-Tyr cross-link (Nackerdien et al., 1991b). The Thy-Tyr cross-link was also produced in chromatin samples, which were treated with H,O z in the presence of Fe(III) or Cu(ll) ions, whereas other DNA.protein cross-links were not produced. The formation of the Thy-Tyr cross-link by HzO z was affected differently by various forms of Fe(lIl) and Cu(II). Fe(III)-NTA, Fe(III)-EDTA or unchelated Cu(ll) were very effective in producing cross-linking, whereas unchelated Fe(III) and NTA- or EDTA.chelated Cu(II) were not. The patterns of the yields of the Thy-Tyr cross-link were analogous to those of the yields of modified DNA bases in chromatin treated under similar conditions. As an example, Fig. 2 illustrates the yields of the Thy-Tyr cross-link in isolated chromatin, which was treated with the H202/Cu(II) system.

Oxygen appeared to be also the main factor in

336

|

u

c

o o

1

3b

E gg

1 2 3 4 s s 7 e o to 11

Fig. 2. Yields of the Thy-Tyr cross-link in isolated chromatin. h chr; 2: chr/Cu(II); 3: c h r / H : O z/Cu(II) ; 4 : c h r / H 2 0 2 / Cu(il)/asc; 5: chr /H 2 0 , / C u ( l l ) / a s c / S O D ; 6: chr /HzO 2 / Cu(ll)/asc/mannitol; 7: ch r / H ,Oz / Cu ( i l ) / a s c / DMSO; 8: chr /H. ,O. /Cu-NTA: 9: chr /HeO, /Cu(I I ) -NTA/asc ; 10: ch r /H .O z /Cu( l l ) -NTA/asc /SOD; I h chr /H 20 , /Cu( l l ) - NTA/asc/DMSO. Graphs represent the mean+SD from three independent experiments. (From Nackerdien et al,,

1991b.)

inhibition of formation of the DNA-protein cross-links other than the thymine-tyrosine cross- link under the conditions of H~O,/metal ion treatment. Generally, oxygen reacts with carbon-

centered radicals at diffusion-controlled rates (for a review see yon Sonntag, 1987), and thus inhibits cross-linking (dimerization) reactions of radicals. The thymine-tyrosine cross-link is thought "~a result from addition of the "OH-generated a11yl radical of thymine to carbon-3 of tyrosine followed by oxidation of the resulting adduct radical and/or from combination of the allyl radical of thymine with "OH-generated tyrosine radicals (Dizdaroglu et al., 1989). Oxygen may not be able to react with thymine or tyrosine radicals prior to cross-linking. Partial inhibition of cross-linking by mannitol and DMSO is consistent with the idea of site-specific generation of "OH upon reaction of chromatin-bound metal ions with H 2 0 2 (Samuni et al., 1983; Ward et al., 1985; Goldstein and Czapski, 1986). Thus, "OH may be generated near thymine and tyrosine molecules in ehro- matin so that mannitol and DMSO may be un- able to completely scavenge "OH.

DNA damage in chromatin of cultured mammalian cells

DNA base damage in chromatin of "y-irradiated cultured cells

in the past decades, ionizing radiation-induced damage to DNA bases in cells has been invosti-

"FABLE I

YIELDS OF DNA BASE PRODUCTS (MOLECULES/10 s DNA BASES) FORMED IN CHROMATIN OF 7-IRRADIATED CULTURED HUMAN CELLS

Product Radiation dose

control 42 Gy 116 Gy 214 Gy 420 Gy

5-OH-5-Me-Hyd 2.91 ± 0.38 4.19 + 0.64 3.04 ± 0.45 2.78 + 0.32 3.07 4. 0.39 5-OH-Hyd 10.40 ± 1.38 - 15.74 4.1.73 * 17.504. !.44 * 23.234-2.88 * 5-OHMe-Ura 0.77±0.09 1.28±0.22 1.89 ±0.51 * .'?.21 4.0.19 * 2.85 +0.35 * 5-OH-Ura 0.38 ± 0.05 0.76 ± 0.14 * !.08 4. 0.19 * 1.59 4- 0.27 * 1.77 ± 0.06 * 5-OH.Cyt 2.44 ± 0.38 3.03 ± 0.59 3.01 ± 0.22 3.26 4. 0.5 ! 4.67 4- 0.54 * Thy glycol 1.63 ± 0.22 3.65 ± 0.51 * 6.56 4. 0.61 * 6.72 4. 0.96 * 10.24 ± 0.83 * 5.6.diOH-Cyt 0.32+0.09 1.15±0.08 * 1.89±0.22 * 2.78+0.11 * 4.134.0.86 * FapyAde 3.26±0.51 4.48± 1.18 6.98±0.99 * 8.33 4.0.67 * 10.024.0.16 * 8-OH-Ade 2.98 ± 0.58 4.51 ± 0.99 6.08 4. 0.64 * 4.22 4. 0.74 5.54 4. 0.77 * 2-OH-Ado 1.95 ± 0.27 3.03 ± 0.10 * 4.42 ± 0.38 * 3.84 4. 0.64 * 4.86 4. 0.58 * FapyGua 2.564-0.48 5.34 ±ll.54 * 14.91 4.3.33 * 20.90± 2.08 * 34.244.1.60 * 8-OH-Oua 7.71 ± 1 . 1 8 12.544.2.43 15.744.2.24 * 15.58± 1.22 * 23.304. 3.07 *

Each value represents the mean +standard error from five independent experiments. * Significantly different from the value in column 1 (p < 0.05). (From Nackerdien et a l . 1992.)

gated extensively. Various measurement techniques have been used to isolate, identify and quantitate a number of modified DNA bases in cells (Hariharan and Cerutti, 1972; Mattern et al., 1975; Frenkel et al., 1981, 1985; Leadon and Hanawalt, 1983; Teebor et al., 1984; Breimer and

Lindahl , 1985; Patil et al., 1985; Kasai et al., 1986; Furlong et al., 1986; Leadon, 1990). Gener- ally, these studies have been limited to identification of one modified base or a small number of modified bases at a time. No specific structural evidence has been provided for the identified products because of the limitations of the analyti- cal techniques used. Recently, we have investigated the formation of pyrimidine- and purine- derived lesions in chromatin of 1,-irradiated cultured human cells (Nackerdien et al., 1992). Twelve modified DNA bases were identified in chromatin samples from cells irradiated at five different radiation doses as well as in those from unirradiated cells. Modified bases and their amounts in control chromatin and in chromatin from irradiated cells are shown in Table 1. Signif- icant increases in the yields of a number of modified bases in chromatin over background levels were observed at a dose as low as 42 Gy. The yields of 5-OH-Hyd, Thy glycol, 5,6.diOH-C'yt, FapyGua and 8-OH-Gua were increased by in- creasing doses of radiation up to 420 Gy, and those of 5-OHMe-Ura, 5-OH-Ura and FapyAde

337

up to 214 Gy. At 420 Gy, no further significant increase in the yields ot these modified bases was observed.

The results showed the formation of a number of pyrimidine- and purine-derived DNA bases in chromatin of 1,-irradiated cells. The yields of modified bases were lower than those in isolated chromatin, which was ~/-irradiated in air-saturated aqueous suspension (Gajewski et al., 1990). Previ- ously, isolated chromatin has been reported to be more susceptible to ionizing radiation-induced damage than chromatin in cells (Roti Roti et al., 1974; Mee et al., 1978; Heussen et al., 1987; Chiu et al., 1992). Modified bases identified in chromatin of ~,-irradiated cells are known to be the typical "OH-induced products of DNA bases (for reviews see yon Sonntag, 1987; Halliwell and Aruoma, 1991). Hydroxyl radical is known to cause = 70% of radiation-induced lethality and DNA damage in oxic cells (Roots and Okada, 1972, 1975; Chapman et al., 1973). Ionization of DNA bases caused by the direct effect of radiation may also account in part for the formation of modified bases (for reviews see Steenken, 1989; Angelov et al., 1991). The yields of guanine-derived bases amounted to = 45% of the net total yield of modified bases. This may indicate the high reactivity of "OH toward guanine residues in cells. Product yields approached a plateau at high radiation doses, The deviation of yields from lin-

m

U ~ . C < 4o.

tTti

2W'

100'

3 .

J 10- 0 I e o

A B C 0 E F • H | J

Fig. 3. Modified DNA bases and their yields in chromatin of H,O2-treated cultured mammalian cells. [3, untreated; ~, treated with 2 mM H202; II, treated with 20 mM H202. A: 5.hydroxy-5-methylhydantoin; B: 5-hydroxyhydantoin: C: 5- (hydroxymethyl)uracil; D: cytosine glycol; E: thymine glycol; F: 5,6.dihydroxycytosine; G: 4,6.diamino-5-formamidopyrimidine; H: 8-hydroxyadenine; I: 2,6.diamino.4.hydroxy-5-formamidopyrimidine; J: 8.hydroxyguanine. Graphs represent the means + SD from

four independent experiments. (From Dizdaroglu et al., 1991a.)

338

earity may result from hypoxic conditions generated by radiation consumption of oxygen. As previous in vitro studies indicate, the presence of oxygen enhances the yields of most of the DNA base products listed in Table 1 (Gajewski et al., 1990).

DNA base damage in chromatin of H202-treated cultured cells

DNA base damage produced in chromatin of H202-treated cultured mammalian cells was studied (Dizdaroglu et al., 1991a). Ten modified DNA bases were identified and quantitated. Fig. 3 illustrates their yields at two concentrations of H202. The amounts of 5-OH-5-Me-Hyd and 5- OH-Hyd observed in untreated cells were not increased by treatment with 2 mM HeO 2. The increase of product yields over background levels depended on the product type. With 20 mM H,O 2, the yields of 5,6-diOH-Cyt, FapyAde and FapyGua were increased = 10-fold over the background levels. Approximately 2.5-fold and 5-fold increases were observed in the yielcis of the other products. The pattern of products suggests the involvement of 'OH in their formation. Hy- droxyl radical may be generated in vivo in rcac- tions of H20 z with chromatin-bound transition metal ions (Haber-Weiss reaction) in a site- specific manner (Samuni et al., 1983; Ward et al., 1985; Goldstein and Czapski, 1986). There is evidence that 'OH plays a role in biological effects of H20 2 in rive involving naturally occurring metal ions (Mello-Filho et al., 1984; Ward et al., 1985; Meneghini, 1988). Hydrogen peroxide alone does not produce the herein identified base products in DNA (Aruoma et al., 1989; Blakely et al., 1990).

DNA.protein cross-links in chromatin of 3" irradiated or H2Oz.treated cultured cells

Formation of DNA-protein cross-links in cultured human cells upon exposure to ,/-irradiation or to HaO, was investigated (Olinski et al., 1992). Chromatin was isolated from exposed and control cells, and then hydrolyzed with HCI and derivatized. Subsequently, typical ions of the trimeth- ylsilyl derivatives of DNA base-amino acid cross. links known from previous in vitro studies (see above) were monitored during GC/MS-SIM

analyses of derivatized hydrolysates of chromatin samples. Among the DNA-protein cross-links known from previous studies, only the formation of the Thy-Tyr cross-link was observed. In 3"- irradiated cells, a linear dose-yield relationship was obtained in the dose range from 8.7 to 82 Gy. At higher doses (up to 400 Gy), the yield approached a plateau and did not increase significantly over the level at 82 Gy. This deviation from linearity is in agreement with previous find- ings that the yields of DNA-protein cross-links in mammalian cells increase linearly with radiation dose in a limited range of radiation doses (Cress and Bowden, 1983; Oleinick et al., 1987). This phenomenon suggests that only limited amounts of thymine and/or tyrosine may be available for cross-linking in cells.

In H202-treated cells, the amount of the Thy- Tyr cross-link increased between ~-2-fold and = 4-fold over the background level in the concentration range from 0.5 to 10 mM of H20, (Olin- ski et al., 1992). At concentrations above 10 raM, the production of the Thy-Tyr cross-link approached a plateau. DMSO and o.phenanthro- line afforded a partial inhibition of cross-link formation. Pretreatment of cells with ascorbic acid or KCN slightly increased the yield of the Thy-Tyr cross-link.

Mechanisms for the formation of the Thy-Tyr cross-link in cells may involve the addition of the 'OH-generated allyi radical of thymine to tyrosine or the combination of the allyl radical of thymine with 'OH-generated tyrosine radicals, as was proposed above for in vitro formation of this DNA-protein cross-link. In 3'-irradiated cells, the allyl radical of thymine may also be produced by deprotonation of ~he thymine radical cation formed by ionization of thymine (Deeble et at., 1990). Similar to the results obtained in in vitro studies, oxygen does not appear to interfere with the formation of the Thy-Tyr cross-link in cells. Partial inhibition of cross-linking by DMSO in H202-treated cells indicates the site-specific nature of cross-linking and the role of "OH. The two mechanisms described above for cross-linking of thymine and tyrosine in 3'-irradiated cells, which involve a radical addition reaction or a radical-radical combi,~ation, may also occur in H2Oa-treated cells. In this case, however, the

339

thymine radical cation is not expected to be formed. Furthermore, the site-specific formation of one thymine radical and one tyrosine radical in close proximity to each other is likely to result from a mechanism different from that described above for irradiated cells. Cyclic redox reactions involving chromatin-bound metal ions, 0 2 and H20 2 may occur giving rise to multiple production of "OH, and thus to multiple hits (Samuni et al., 1983; Ward et al., 1985). Because of the site specificity of these reactions, oxygen may not prevent DNA-protein cross-linking between thymine and tyrosine.

DNA base damage in chromatin of animal organs in vivo

Ni(II) salts are known to be carcinogenic to humans and animals (for a review see Costa, 1991). However, the mechanisms involved in Ni(ll)-mediated carcinogenesis remain elusive. Recently, Ni(lI)-m~diated oxidative DNA base damage has been studied in chromatin of kidneys of pregnant female rats and their fetuses (Kaspr- zak et al., 1992). Treatment of animals with Ni(ll) salts was done under the conditions leading to initiation of sodium barbital-promotable renal tu- mors in rats exposed to Ni(ll) salts either directly or transplacentally. Renal chromatin was isolated from control animals and from treated animals and their offspring. Chromatin samples were subsequently analyzed by GC/MS-SIM after hydro- lysis and derivatization. Eleven modified DNA bases were identified. Upon treatment of animals with Ni(II) salts, the amounts of modified bases in their renal chromatin were significantly increased over the levels observed in renal chro. matin of control animals. The same effect was observed in renal chromatin of fetuses of Ni(ll)- treated pregnant female animals. The pattern of DNA base products indicated a possible involvement of "OH in their Ni(ll)-mediated formation. The modification of DNA bases in renal chromatin may constitute initiating events in Ni(ll)-induced carcinogenesis.

Conclusions

A variety of free radical-producing systems produce numerous pyrimidine- and purine-de-

rived DNA base modifications and DNA-protein cross-links in mammalian chromatin in vitro and in vivo. Evidence indicates that most l:roducts arise as a result of reactions of "OH with DNA. In the case of ionizing radiation, other radical species, e~q and H atom also cause some DNA modifications. The types and quantities of the DNA modifications profoundly depend on the free radical-producing system. Substantial quantitative differences exist between the effects of the metal ions when present in these systems. The presence of oxygen favors the formation of the majority of DNA base products. On the other hand, some base products and most DNA-protein cross-links are generated only in the absence of oxygen. The contribution of the DNA modifications in mammalian chromatin to the biological effects of free radical-producing systems is as yet unknown. In the past, several modified DNA bases have been investigated for their biological consequences and some of them have been found to be associated with mutagenesis (Wallace, 1987; Shirnam6-Mor~ et al., 1987; Hayes et ai., 1988; Basu et al., 1989; Wood et al., 1990; Moriya et al., 1991; Guschlbauer et al., 1991; Cheng et al., 1992). The variety of free radical-induced DNA modifications in mammalian chromatin hampers the assessment of their role in mutagenesis, carcinogenesis and aging. The chemical characteri- zation and quantitation of such modifications should form the basis of farther studies for the understanding toward their biological significance.

Acknowledgement

Research in the author's laboratory is sup- ported in part by the Office of Health and Envi- ronmental Research, Office of Energy Research, Department of Energy, Washington, DC.

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Oxidative damage to DNA in mammalian chromatin