Changes in DNA supercoiling in fibroblasts cultured in the presence of hydralazine

Biochimie (1998} 80, 627-630 © Soci6te franvaise de biochimie e| biologic m~K'culan'c / Eb,e~ ie~'. Paris

Changes in DNA supercoi[ing in fibr blasts cuRured in the presence of hydralazine L u d m i l a We,g l a r z '~, A n e t a K o c e v a - C h y t a b* '~Dep,~~m~m of Biochemistry a~d Chemisto; Silesian Med&~d Academ3; Medvk6w 18. 40-752 Katowice, Pola~M ~'l)el,anment of Thermobiology, Univerxisv of L,'MS~. Bamwha 12/16. 90-237 L6dL Poland

(Received 23 October 1997: accepted 2 April 1998) Ahstraet .........We have analyzed changes in tile supercoiling of nucleokl DNA of murine fibroblasts cultured in the presence of hydralaeine, The entire DNA attached t~ the nuclear roan'ix ~as extracted from the cells and scdimenled in neutral sucrose density gradients co|ltahlhlg cthidium bromide, Nucleoids fi'om cells treated with hydralazme responded to increasing ethidium bromide concentrations in a different way than those from control cuhures. That is, supercoiled loop,,, of DNA unwomld with lower concentrations of ethidium bromide sedflnented less rapidly than those of control cells, indicating that hydndazine reduced the degree of DNA supercoiling. Also, nucleoids from the drug-treated cells resis|ed the transition from rela×ed to positive supercoilmg at higher concentrations of ethidium bromide, Changes in nucleoid DNA supercoiling ton'elated direc:l,, ~ith the dose of hydralatme m the fibroblast culture. Q St~ciOlefrangaise de biochirnie el biologic molOculan'e / Elsevier, Paris

DNA supercoiling I ethidium bromide / nucleoid / hydralazine / fibroblasts I. Introduction Hydralazine, an aromatic hydrazine derivative has becn widely used in the treatment of severe hypertension II, 2 I. Its use, however, may be associated with the induction of an autoimmune syndrome resembling systemic lupus erythematosus iSLE)11, 3, 41. Various methods have revealed that SLE patient's sera contain antibody against DNA 14-61. Drugoinduced inlnmnc reactions are thought to be caused by reactive metabolites of the offending agents 17, 81, and hence, the metabolism of hydralazine may be of importance in the patlmgenesis of hydralazin¢ induced lupus, Several researehcr~ have investigated the metabolism of hydralazine in various systems and prco sented evidence lbr the Iormation of the reactive interme° diates that might have a role in hydra!azine°metliated idiosyncratic reaction 19-121. I/ydralazine was shown to t,e radicals and m species be oxidized enzytnalically to I"e, capable of binding to micmsomai or leukocyte protein II 3-151. Moreover, Ii"ee radicals or the derived active species generated during metal- or peroxidase-catalyzed oxidation of hydralazine participated in site-specitic cleavage of isolated DNA 1161. Hydralazine alone caused degradation of spin-labeled DNA that was augmented in the presence of Fe(ll), as measured by changes in the ESR spectra ll71. The drug also increased the production of thiobarbituric acid reactive products from DNA 1181. Similar results were observed by Weglarz and Bartosz 1171. Hydralazine-induced DNA damage may bc

Abbreviations: DNA, deoxyribonucleic acid; ESR, electron spin

remnancc; EDTA, ethylenediaminetetraacetic acid.

relevant Ibr tile expression of the toxic properties of hydralazine. The fact that antibodies to chemically modilied DNA are induced is of interest in relation to hydralazine-induced SLE 119, 201. Antibodies to DNA with unusual conformation were observed in patients with SLE 121,221. The interaction of Ilydralazine with DNA has been analy/cd in the cxtracellular systems using DNA derived from wu'ious sources. Analysis of damage to the hlghel'order I)NA structure in the nucleus, rathc~ than ,just |he DNA itself, may provide addilhmal insight into the toxic eVOlilS that occur when ccll~ al'e exposed m some d|'ug+,, illehtdiilg hyth'al~t,eitlc. It is possible lo ¢xa|/!i!!¢ Ihc higher,order DNA structure Csupercoils) by extracting fronl cells DNA still attached to the nuclear matrix, and unwinding the negatively supercoiled loops with ethidium bromide 123, 2,11. In this study we have analyzed the organization o1' supercoiled DNA in tibalblasts cultured in vitro in the presence of hydralazine, qb achieve this, we have examined the effect of ethidium bromide intercalation on DNA supercoiling within tibroblast nucleoid structures. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation pmliles in 15=3(Yh. neutral sttcrosc gradients.

2. Materials and methods The experiment was carried out on primary cultures of Balb 3"I"3 mouse libroblasts isolated from tile liver by trypsinization 1251. The cells were cultivated in Eagle's tninimum essential medimn supplemented with 5~ heatinactivated fetal call" serum, penicillin (100 U/ml,) and streptomycin 120 ~tg/ml,). Cells were kept at 37 °C in a

628 water-saturated incubator containing 5% CO2 and 95% air and were monitored to ensure they were mycoplasma-free throughout the period of use. Cells were cultured in the presence of four concentrations of hydralazine (l-hydrazino-phtalazine, Sigma) as follows: 25, 50~ 75 a n d l ~ p ~ m L of the culture medium. nedium and [0.22 Bin) at 24 h after starting the culture. Control cells were cu~. "'ed in parallel without the drug. After 72 h of hydrala~::ine treatment the cells were washed with phosphate-buffered ~line (PBS)to remove non-adherent cells and trypsinized for a period of up to 3 rain to harvest the monolayer. The technique used tbr preparation of nucleoids and their contrifugation was based on the original description by Cook and Brazel11231, Sucrose gradients (! 5-30%, pH 7,5) were prepared in centrifuge tubes using an lSCO density gradient former, They contained 2 M NaCI, 0.01 M Tris, 0,01 M EDTA and variable concentrations of ethidium bromide (I, 3, 5, I 0 and 20 ~g/mL) (EB, Sigma). The lysis solution contained 2 M NaCi, 0.01 M Tris (pH 8,0), 0,2 M EDTA and i % Triton X. 100, and 150 pL of it was carefully layered on top of the ~radienl, A 50 pL suspension of dispersed cells (!.5 x !0" to 3 x 10" cells in PBS) was applied on top of a lysis mixture, The cells were allowed to lyse for I h in the dark at room temperature, Afterwards gradients were spun at 15000 rpm for 2 h at 20 °C in a Beckman model L5-65 ultracentrifuge, The position of the nucleoids in the gradients was determined by the fluorescence of the gradient Ii'actions which were collected from the top with the use of an ISCO gradient fractionator, Fluorescence measur,.~ments were made using a JobinoYvon spectrophotofluoromettn with excitation at 360 nm and emission at 590 nm, Each centrifugation run included a tube of control cells sedimenting in a sucrose gradient containing no EB, which served as a rcl~rence, The position of lhe control nucleoids in the ~radient lacking EB was determined after adding the intel~alating dye at a final concentration of 20 mg/mL to the fn~ctions b~i~e d'~e fluorescence measurements ~ere takers, 3,

Wqglarz and Koceva.Chyla 100

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traveled by the control nucleoids sedimenting in the absence of EB under the same conditions. A ratio of 1 refers to the control nucleoids sedimenting in gradients containing no EB (figure I). Hydralazine, when incubated with fibroblasts, affected chromatin structure so that isolated nucleoids sedimented less rapidly in neutral sucrose gradients than those from control cultures {figures I ~ 3). Intercalated ethidium bro, mido affected control nucleoid sedimentation in a biphasic manner, as shown by the rues! upper curve in/igtm, 3, The biphasic expansion and contraction of nucleoids in response to edlidium bromide titration was due to the relaxati~m of

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Fluo~scenc¢ pretties of the sucrose density ~radients :ontaining difie~nt concentrations of elhidium bromide and reflecting sedimentation of nucleoids released from control fibroblasts and from fibmblasts cultured in the presence of the highest concentrations of hydralazmc, that is I ~ Bg/mL, are shown in fi,quP~t,s I and 2 res~ctively, The changes in sedimentation rate of nucleoids i?om tibroblasts as a function of ethidium bromide ,'oncentra. ties within sucrose gradients are presented In,ligu~. 3, The distance the nucleoid traveled down the gradient was detemfined by measuring the distance lYom the meniscus to the nucleoid peak in the gradient (determined fn~m the fluorescence measurements). The distance traveled by nucleoids in gradients containing different concentrations of EB wan expressed as a ratio relative to the distance

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Figure 2. Fluorescenceprofiles of the sucrose density gradients containing uucleoids released t'mnl libmblasts treated with I00 pgtmL of hydralazinc,

Changes in DNA supercoiling in fibroNasls cultmed in the prcsc~ce of hydrak~gnc 10

629

elhidium bromide indicated that nuc~coids fai~cd to bc condensed ~comracted} to the cxtem the comrol audeoids did.

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Figure 3. Effect of ethidium bromide on .,,cdimcntationdistance o1" fibroblast nucleoids in neu|ral sucrose gradients,

tions and the subsequent imposition of positive rewinding at higher EB concentrations. "['he effect of ethidium bromide was modilied by hydralazine in a way lhat is consistent with a reduction of the degree of DNA supercoiling. Decreased sedirnentation rates of the nucleoids Ibllowing hydralazinc treatment, in the presence el" the EB concentrations up to 5 tng/mL relative to the controls indicated that the nuclcoids exhibited decreased DNA negative superhelicity. As the dose of hydralazinc in the cell culture increased, the sedimentation rate of nucleoids continuously decreased. Also the mininmm sedimentation rate at the relaxing elhidium bromide concentration decreased when the drug concentration increased. The concentration of ethidium bromide which minimized the sedimentation ntte was the same (5 ttglml,) for nucleoids from both the controls and the cells treated with hydralazine. At the higher ethidium bromide conceno trations (> 5 pg/mL} the sedimentation behavior of nucle° oids l~om hydralazine treated cells was totally different from that of control nucleoids (ligure 3). Whereas these concentrations (i.e., from 5 gg/mL to 20 [tg/mL) of the dye caused the relaxed DNA molecules li'om control cells to coil back Ibllowed by an increase in nucleoid sedimentation rate, they had little eITeet on the sedimentation of nucleoids from cells exposed to hydralazine. Thus, they caused a monotonous and slight increase in the rate of sedimentation of nucleoids from cells treated with the two lower (i.e., 25 t,tg/mL and 50 tug/mL) hydralazine concentrations and they hardly affected the rate of sedimentation of nucleoids derived from cells cultured in the presence of the two higher concentrations o1" hydnflazine (i.e., 75 pg/mL and 100 pg/mL). Much shallower biphasic curves produced by nucleoids in response to titration with

Spatial organization of DNA in cflromafin consi,,~s in the arrangement of DNA into loop domains [231 which are anchored to the nuclear matrix [24, 26[. The higher order chromatin structures may be examined by extracting nuclei from cells using buffers that remove most stabilizing proteins [24, 27]. Such nuclei, termed nucleoids, retain many of the morphological features of nuclei, and their sedimentation rate in sucrose gradients reflects the real nuclear morphology [281. The sedimentation rate of nucleoids in neutral sucrose gradients is determined by nucleoid mass and the compactness of tile DNA loops or domains 12~.~I. We have used the nucleoid sedimentation technique to analyze changes in chromatin structure of fibroblasts caused by hydralazine. The degree of supercoiling in nucleoid DNA was monitored by sedimentmg nucleoids in ncatral sucrose gradient containing ethidium bromide. In the data presented here, nucleoids from the control cells responded to increasing ethidium bromide concentrations in at way clulracteristic of the change from negative to positive supercoiling. No similar imposition of positive supercoiling was observed in nucleoid DNA following hydralazine treatment. Alterations in supercoil compaction may result primarily from the loss of topological corn straint on the DNA loops due to insertion of single-strand breaks in the DNA [23, 27 [. The rewinding of DNA loops was impaired in a hydra!azine dose.dependent manner rcliccting an increase in the number of affected super° coiled loops. The resuhs in Jigtoe 3 indicate tile different sedimcn ration distances lot the nucleoids released from hydrala/ille treated awedcontrol ceils at the all of l£B co,;centra. lions applied. That is, the scdimcntatiott Lat¢:; weru less for the nucleoids from hydralazine treated fibroblasts. Also the decrease in sedimentation rote at the relaxing ethidium bromide concentration compared to the sedimentation rate in the absence of EB was less for the nucleoids l?om hydralazine treated cells than lbr nucleoids from control cells, it means that: i) there was a loss of DNA negative superhelicity in nucleoids l'rom hydralazine treated cells due to the DNA strand breaks; ii) nucleoids from these cells contained fewer domains with DNA negative super° helicity than control fibmblasts and thus showed less of a decrease in sedimentation rate at the relaxing ethidium bromide concentration, it cannot be excluded that also other changes induced by hydralazine within chmmatin structure might alTcct the sedimentation behavior of nucleoids e.g., hydralazine could alter aflinity between supercoiled looped DNA and the nuclear matrix producing l'ewcr anchor points and decreasing the nucleoid mass. This factor could also contribute to the maximum expansion of the nucleoids Ii"om hydralazine treated cells. Of

630

Wgglarz and Koceva-Chyla

possible relevance to the changes in nuclear matrix core size affecting the ~dimentation rate are the results of the studies on the nucleoids isolated from irradiated ataxiatelangiectasia fibroblasts I301. As shown in figure 3, nuclcoids from hydralazinetreated cells exhibited maximum relaxation at the same ethidium bromide concentration as nucleoids from control cells. This sedimentation pattern is typical for the nucleoids with strand breaks which tend to exhibit minimal sedimentation at the same ethidium bromide concentration as control 127, 311. In contrast, nucleoids with decreased DNA negative superhelicity and intact DNA exhibit minimal sedimentation at a lower ethidium bromide concentration than control 1311. These data indicate also that in both kinds of nucleoids the DNA domains that were negatively supercoiled had approximately the same average superhelical density. A similar observation was noted by others 1311 who analyzed the ethidium bromide titration of nucleoids derived from two different fibroblast populations. Both kind of cells differed in the amount of DNA strand breaks and exhibited minimum sedimentation rate at the same concentration of the dye. The present results show that the DNA damage caused by hydralazine leads to the alteration of DNA conformation and superhelical properties. Such changes in the DNA higher order structure may enhance the immunogenic potential of DNA followed by the formation of antinuclear antibodies, Unusual conformations of DNA are known to be able to induce antibodies to DNA 121,221. tilus the altered structures of chromatin components induced by hydralazine might be of interest in relation to hydnllazineoinduced lupus,

191 Hofstra A.H., Metabolism of hydralazine: relevance to druginduced lupus, Drug Metab. Rev. (1994) 26 485-505. I10t Sinha B.K., Matten A.G.. Oxidative ntetabolism of hydralazine. Evidence for nitrogen centered radicals formation, Biochem. Biophys. Res. Commun. 105 (1982) i 044-105 i. 1111 Sinha B.K.. Enzymatic activation of hydralazine derivatives. A spin-trapping study, J. Biol, Chem. 258 (1983) 796-801. 1121 Weglarz L., Bartosz G., Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts, Free Rad. Biol. Med. I1 (1991) 149-155. 1131 Lacagnin LB.. Colby H.D., Dalai N.S., O'Donneli J,P., Metabolic activation of hydralazine by rat liver microsomes, Drug Metab. Dispos. 14 (1987) 549-554. il41 Hofstra A.H.. Matassa L.C.. Uetrecht J.P., Metabolism of hydralazinc by activated leukocytes: implications for hydralazine-induced lupus. J. Rheumatol. 18 11991) 1673-1680. I151 Hofstra A.H., Uetrecht J,P.. Reactive intermediates in the oxidation of hydralazine by HOCh the major oxidant generated by neulro~ phils, Chem. Biol. Interact. 89 (1993) 183.196. [16l Yamamoto K., gawanishi S., Free radical production and sitespecilic DNA damage induced by hydralazine in the presence of metal ions or peroxidase/hydrogen peroxide, Bk~hem. Pharmacol. 41 (1991) 905-914, [ 171 Weglarz L.. Bartosz G., Spin label detection and I've radical nature of DNA damage by hydralazine. Int. J, Biochem. 23 (19911 663 -667. 1181 Sinha B.K.. Patterson M.A., Free radical metabolism of hydralazinc. Binding and degradation of nucleic acids, Biochem, Pharmacol. 32 (1983) 3279,3284. 1191 Biount S., Grifliths H.R,. Lunec J,, Reactive oxygen species induce antigenic changes in DNA, FEBS Lett. 245 (1989) 10(}-i04. 1201 Blount S., Lunec J., Grifliths H.. Herbert K.. Isenberg D., Binding of anti-DNA antibodies to oxidatively damaged DNA in spouses and relatives of patients with systemic lupu~ erythematosus, Imlminol. Left. 41 (1994) 135°138. 1211 Lurer E,M,, M611er A,, Nordheim A.. Stellar B.D.. Rich A., Antibiotics spccilic li~r Icfldlanded Z~DNA, Proc, Nail, Acad. Sci, USA 78 {19811 3546~3550, 1221 Lal~r !~,M,, Val!e R,T,C,, M611er A,, Nordhelm A,, Schur P,It., Ricl! A,. Stellar B,D,, Zl)NA-sl~'cilic antibodies in hunlan sys o

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