Original Contribution

CYTO- AND GENOTOXIC EFFECTS OF NOVEL AROMATIC NITROXIDERADICALS IN VITRO

ELISABETTA DAMIANI ,* L UCEDIO GRECI,* and PATRIZIA HRELIA†

*Dipartimento di Scienze dei Materiali e della Terra, Universita`, Ancona and†Dipartimento di Farmacologia,Universita, Bologna, Italy

(Received4 August1999;Revised4 November1999;Accepted9 November1999)

Abstract—Because of the increasing interest in the use of nitroxide radicals as antioxidants and probes for variousapplications in biological systems, the question of their toxicity is of paramount importance. Cytotoxicity andmutagenicity studies have been extensively performed with the commercially available aliphatic nitroxides, and thegeneral outcome is that these compounds are nonmutagenic and relatively noncytotoxic. In this study, the cytotoxicityand genotoxicity of a new class of aromatic nitroxides that we have synthesized (i.e., indolinonic and quinolinicnitroxides), whose antioxidant activity has been established in both chemical and biological systems, were evaluated andcompared with those of two commercial nitroxides and with that of butylated hydroxytoluene (BHT). The mutagenicityassay was performed usingSalmonella typhimuriumtester strains TA98, TA100, and TA102, chosen on the basis oftheir ability to detect various types of mutations and their sensitivity to oxidative damage. None of the compounds testedwere found to be mutagenic. The colony-forming assay (CFA) using Chinese hamster ovary (CHO) AS52 cells wasemployed for determining the cytotoxicity of the test compounds. On comparing the effective dose that inhibits the CFAby 50% (IC50), most of the compounds tested on an equal molar concentration basis were less toxic than BHT.Therefore, the overall results obtained correlate well with the data reported in the literature on the toxicity of aliphaticnitroxides and lend support to the possible use of these compounds as therapeutic antioxidants. © 2000 ElsevierScience Inc.

Keywords—Aliphatic and aromatic nitroxides, Antioxidants, Mutagenicity, Ames test, Cytotoxicity, CHO/AS52 cells,Free radical

INTRODUCTION

Cyclic nitroxides (alternatively termedaminoxyls) are aunique group of compounds bearing an unpaired electronon the N-O function, which are included in an aliphaticor aromatic ring system [1,2]. They are widely exploitedas biophysical probes and labels for membrane and pro-tein studies because of the sensitivity of their electronparamagnetic resonance spectra to the microenvironmentand their mobility. An additional contributing feature istheir remarkable stability and persistence as free radicals,which is mainly a result of delocalization of the unpairedelectron between oxygen and nitrogen, explained interms of a lowered ground state energy of the three-

electron nitrogen-oxygen bond system for aliphatic ni-troxides or of delocalization of the unpaired electroncloud over the conjugated system attached to the nitro-gen in the case of aromatic nitroxides. Also of majorimportance is the absence of dimerization and dispropor-tionation [1,2]. These factors explain why nitroxide spinlabels have been important biophysical tools for under-standing the structure of membranes, proteins, and otherbiopolymers [3] since they were first introduced by Mc-Connell in 1965 [4]. Besides their use as biophysicalprobes, they have also been employed as contrast agentsfor nuclear magnetic resonance imaging [5] and are usedin electron paramagnetic resonance imaging [6]. Further-more, studies have demonstrated that the incorporationof a nitroxyl moiety into the chemical structure of anti-cancer medicines [7] as well as simultaneous injection ofnitroxide radicals with cytostatic agents [8] can decreasethe acute toxicity exerted by anticancer drugs.

Address correspondence to: Prof. Lucedio Greci, Dipartimento diScienze dei Materiali e della Terra, Universita`, Via Brecce Bianche,I-60131 Ancona, Italy; Tel:139-0712204408; Fax:139-0712204714;E-Mail: greci@popcsi.unian.it

Free Radical Biology & Medicine, Vol. 28, No. 3, pp. 330–336, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

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In the past decade, however, the use of nitroxideradicals in biological systems other than as spin labelsand contrast agents has greatly expanded. In fact, there isnow an accumulating body of evidence indicating thatnitroxide radicals protect a number of biological systemsat the molecular, cell, organ, and whole-body levels fromthe oxidative stress inflicted by diverse insults [9,10]. Forexample, Samuni and his colleagues [11,12] have dem-onstrated that nitroxides can act as superoxide dismutasemimics that can protect isolated mammalian cells andcardiomyocytes from oxidative stress. It was also foundthat nitroxides protect cultured mammalian cells exposedto ionizing radiation [13] and microsomal membranesagainst lipid peroxidation [14]; they also protect bacterialcells exposed to hydrogen peroxide, hypoxanthine/xan-thine oxidase [15], cytotoxic drugs such as streptonigrin[16], and the naphthoquinones juglone and menadione[17]. This antioxidant activity, which was first recog-nized almost three decades ago [18], results from theircapability to selectively react with potentially toxic freeradicals such as the superoxide radical that they dismu-tate [11] and the carbon-centered radicals that they trapat the N-O function [19]. In addition, nitroxides oxidizereduced metals, thus inhibiting their participation in met-al-catalyzed, free radical–generating reactions [15,20].

The majority of these studies on the applications ofnitroxide radicals were carried out with the commercial

aliphatic nitroxides, piperidines, pyrrolidines, and oxazo-lidines. Nevertheless, in recent years, our group hasfocused its attention on a new and different class ofnitroxides, namely, the indolinonic and quinolinic aro-matic nitroxides that we have synthesized. These nitrox-ides have been shown to efficiently scavenge all kinds ofradicals. They couple with carbon-centered radicals, giv-ing alkylated hydroxylamines [21,22], and unlike thealiphatic nitroxides, they react with all oxygen-centeredradicals such as hydroxyl [23], alkoxyl [24], peroxyl[25], and aroyloxyl [26] to form nonparamagnetic com-pounds. Thus, the reaction of indolinonic nitroxides withperoxyl radicals leads to the quinoneimine N-oxide7 inhigh yields as shown in Fig. 1. Quinoneimine N-oxidesare the final products even in the reaction with the othermentioned oxygen-centered radicals. Thiyl [27] and ami-nyl radicals [28] are also efficiently trapped by this classof aromatic compounds. In consideration of this out-standing reactivity towards radical species and the typeof radicals produced during oxidative processes, thesenitroxides have been studied in various biological sys-tems ranging from lipids [29–32], proteins [33], andDNA [34,35] to intact cells [36,37] with the aim oftesting their potential as antioxidants. In every case, theywere shown to possess a relatively efficient antioxidantactivity with a marked reduction in the degree of oxida-tion of the systems under study.

Fig. 1. Chemical structures of indolinonic (1–3) and quinolinic (4) nitroxides, the piperidinic nitroxide TEMPO (5), the pyrrolidinicnitroxide TMPO (6), the quinoneimine N-oxide (7), and BHT. The figure also shows how nitroxide1 reacts with peroxyl radicals(ROO•) to give compound7.

331Genotoxicity and cytotoxicity of nitroxide radicals

In view of the significance of nitroxides as potentialpharmaceutic agents and probes for various applicationsin biological systems, the question of the toxicity ofnitroxides is of paramount importance. Cytotoxicity andmutagenicity studies have been extensively performedwith the commercially available aliphatic nitroxides on awide range of experimental models, and the generalconclusion from the data obtained is that these com-pounds are nonmutagenic and relatively noncytotoxic[38–41]. The doses required for lethal effects are high,and no long-lasting effects have been observed in survi-vors of LD50 (the dose of a substance which is fatal to50% of the test animals) studies. There are no dataavailable on the possible cytotoxicity and/or mutagenic-ity of the aromatic indolinonic and quinolinic nitroxidesthat we have synthesized and studied; hence, we thoughtit of interest to investigate their effects on cell survivalusing Chinese hamster ovary (CHO) cells (a widely usedmodel system for mammalian cells in culture) and theirmutagenicity using the Ames test. In this study, thequinoneimine N-oxide, which is the main transformationproduct with oxygen-centered radicals, was also consid-ered as well as the piperidinic nitroxide TEMPO and thepyrrolinic nitroxide TMPO, both of which are commer-cially available and were included for comparison. Wealso thought it of interest to compare all the above-mentioned compounds with butylated hydroxytoluene(BHT), an additive found in many foodstuffs used forpreventing the undesirable effects of oxidative degrada-tion [42].

MATERIALS AND METHODS

Indolinonic (1–3) and quinolinic (4) nitroxides [43,44], quinoneimine N-oxide (7), and TMPO (6) [45] weresynthesized according to the methods described in theliterature (Fig. 1). TEMPO (5) and BHT were obtainedfrom Sigma Chemical Company (Milan, Italy). The iden-tity and purity of the compounds were checked by thin-layer chromatography and by mass spectroscopy on aCarlo Erba (Milan, Italy) QMD 1000 spectrometer. Di-methyl sulfoxide (DMSO) and all other chemicals andsolvents were of the highest commercial purity available.

Mutagenesis assay

The mutagenesis assay was carried out according tothe method of Maron and Ames [46] using theSalmo-nella typhimurium tester strains TA98, TA100, andTA102 (auxotrophs for histidine), which were kindlyprovided by Dr. Bruce N. Ames.

It was ascertained that these strains retained the deeprough character, the plasmid pKM101, and the specific

response to a panel of standard mutagens, namely, 4-ni-tro-O-phenylenediamine, sodium azide, and mitomycinC.

The plate incorporation assay was used in evaluatingthe mutagenic potential of each compound. Fresh cul-tures for each experiment were prepared by cultivatingfrozen permanents in Nutrient Broth Difco (Difco, De-troit, MI, USA) for 16 h at 37°C. The nitroxides weredissolved in DMSO and incorporated with the indicatorstrain (in the resting phase) into an agar overlay.

The nitroxides were tested up to their toxicity orsolubility limit. All plates were set up in duplicate, andall experiments were reproduced at least twice. A treat-ment resulting in a positive dose-response curve with atleast twice the number of revertants per plate above thatobtained in the solvent control was considered to pro-duce a positive mutagenic response. Mutagenic potency,expressed as the mean induced revertant frequency perplate per micromole of the tested compound was ob-tained from the slope of the linear regression line fitted tothe increasing portion of the dose-response curves usinga statistical computer program. The mutagenic potency isan index of the mutagenic strength of a compound as-sayed within a specific strain. Background reversionrates were subtracted before constructing the dose-re-sponse curves. The spontaneous revertant frequencies foreach strain (6 SD) were as follows: TA98, 32 (6 2.01);TA100, 115 (6 11.31); and TA102, 280 (6 27.93).

Cytotoxicity assay

Stock cultures of AS52 cells (obtained from Dr. K. R.Tindall, National Institute of Environmental Health Sci-ence, Research Triangle Park, NC) were maintained inmonolayer in F12 medium containing 5% heat-inacti-vated fetal calf serum and 0.01% gentamycin [47]. AS52cultures were routinely reactivated in F12 medium con-taining 5% dialyzed heat-inactivated fetal calf serum andgentamycin (F12FCS5), which was supplemented with250 mg/ml xanthine, 25mg/ml adenine, 50mM thymi-dine, 3mM aminopterin, and 10 to 20mg/ml mycophe-nolic acid for 2 d. After a 48 h recovery period inF12FCS5 medium, duplicate cultures of;106 cells per25 cm2 flask were treated (day 0) with nitroxides dis-solved in DMSO (1%) for 5 h at37°C in F12 mediumwithout serum. After treatment, cells were washed threetimes with Hank’s balanced salt solution (HBSS) andincubated in F12FCS5 medium. Cytotoxicity was esti-mated 19 to 24 h after treatment by plating an aliquot of200 cells in F12FCS5 in triplicate for each culture. After7 to 8 d, the resulting colonies were fixed, stained with acrystal violet solution (1%), and counted. Inhibition ofcell growth was assessed by evaluating the cloning form-ing ability, a parameter related to basic cellular func-

332 E. DAMIANI et al.

tions, in untreated and treated cultures. Cytotoxicity isexpressed as the percentage of survival relative to that ofthe untreated control cultures. Each test compound stud-ied was tested in at least two separate experiments.

RESULTS AND DISCUSSION

In the present study, the mutagenicity assay was per-formed on highly purified nitroxides1 through 6 andquinoneimine N-oxide7 (Fig. 1). This selection of com-pounds was made primarily because the aromatic nitrox-ides1 through4 and similar derivatives synthesized byus have now been extensively investigated as antioxi-dants in different biological systems. In fact, previousstudies have demonstrated their ability to prevent linole-nic acid oxidation [29] and copper-mediated low-densitylipoprotein peroxidation [30] in a dose-dependent fash-ion. Protein and lipid peroxidation of rat liver micro-somes [31,32] and albumin [33] subjected to differentfree radical insults were also inhibited in the presence ofthese nitroxides. Moreover, these compounds were re-cently shown to protect DNA from damage when illu-minated in the presence of a common sunscreen ingre-dient [35] and to protect DNA and lipids from damage instressed trout nucleated erythrocytes [34]. In addition,hemolysis ofa-loaded human erythrocytes was reducedby these nitroxides [36]. In all these studies, the effectiveconcentration of the nitroxides ranges between 1 and 100mM depending on the system under investigation. Thequinoneimine N-oxide7, which is the main transforma-tion product of the above-mentioned aromatic nitroxideswith oxygen-centered radicals, was also included in thisstudy because it was of interest to see its effects and howit compared with the parent compound. Likewise, thecommercial nitroxides TEMPO and TMPO were alsostudied for comparison. These two aliphatic nitroxideswere chosen as representatives of the most commonlyused nitroxide magnetic resonance imaging contrastagents, anticancer probes, oximetry probes, and antioxi-dants.

In this study, strains ofS. typhimuriumTA98, TA100,and TA102 were used; these strains detect various typesof mutations such as frame-shift mutations (TA98) andbase-pair mutations (TA100) and are sensitive to oxida-tive damage induced by chemical agents or ionizing andultraviolet radiation (TA102). The results obtained onTA98, TA100, and TA102 strains ofS. typhimuriumarereported in Table 1, where the specific mutagenic activityis expressed as revertants per micromole. The com-pounds tested in the concentration range of 0.02 to 5.43mM per plate induced no significant increments in thenumber of spontaneous revertants with all the strainsexamined. Therefore, in these experimental conditions,none of the compounds tested resulted mutagenic. These

results correlate well with data reported in the literatureconcerning other nitroxide derivatives and their meta-bolic products. Sosnovsky [40], who studied four proto-typic aliphatic nitroxides, including TMPO, found littleif any mutagenesis using an Ames-type test system withS. typhimuriumtester strains TA102 and TA104. Otherin vitro assays involving aliphatic nitroxides and theirreduced congeners (i.e. their corresponding amines andhydroxylamines) gave no mutagenic response [48]. John-stone et al. [49] examined the effect of TEMPOL (the

Table 1. Mutagenicity of BHT and Compounds1 Through7 inSalmonella typhimurium

Compound

Concentration Revertants per platea

(mM perplate) TA98 TA100 TA102

1 0.07 306 3 1126 3 3146 20.13 276 2 1126 4 3216 20.27 276 3 966 2 3466 20.80 256 2 956 1 2826 1

rev/mMb 0 0 02 0.02 386 2 996 1 2946 3

0.04 356 2 1126 3 3106 110.08 356 2 1016 5 3876 100.16 386 3 1106 2 2866 3

rev/mMb 0 0 03 0.05 296 2 1266 4 2976 20

0.11 366 3 1176 1 2866 50.21 316 2 1226 6 3366 380.42 366 3 1236 5 2506 71.26 406 3 1806 20 2546 15

rev/mMb 0 0 04 0.02 336 1 1206 1 3496 2

0.04 406 1 1406 1 4036 40.07 386 1 1476 4 3606 220.15 336 1 1326 4 2746 50.29 286 1 1336 8 3356 24

rev/mMb 0 0 05 0.16 376 2 946 3 2366 13

0.32 316 1 1036 4 2656 100.64 306 2 946 3 2396 81.92 276 1 1126 4 2816 15

rev/mMb 0 0 06 0.14 306 2 966 1 3676 2

0.27 266 1 976 1 3596 10.54 306 2 866 8 3636 11.63 286 3 796 6 3526 25.43 296 3 856 2 3416 1

rev/mMb 0 0 07 0.03 286 3 1086 3 3036 4

0.06 366 1 1126 5 2936 20.13 356 1 1346 6 2856 20.26 316 2 1076 9 2306 300.77 366 2 1206 4 2526 27

rev/mMb 0 0 0

a The results are the mean6 SD from two to three independentexperiments. Background reversion rates were subtracted. They were32 6 2 for TA98, 1156 11 for TA100, and 2806 28 for TA102.

b Calculated from the regression line fitted to the dose-responsecurves. No activity (0) was assigned if no mutagenicity was detected upto the highest or bacterial killing dose.

rev/mM 5 mean induced revertant frequency per plate per micro-mole of tested compound.

333Genotoxicity and cytotoxicity of nitroxide radicals

4-hydroxy derivative of TEMPO) on radiation-inducedchromosomal aberrations in peripheral human blood andshowed that at 10 and 50 mM TEMPOL caused nochromosomal aberrations. Moreover, TEMPOL demon-strated antimutagenic activity by preventing the muta-genic effect of bleomycin or hypoxanthine/xanthine ox-idase in CHO cells [13,50]. For this reason, nitroxidesare being investigated as potential antitumorigenic prep-arations, and this antitumorigenic activity is attributed tothe antioxidant activity of nitroxides [9,10]. In anotherstudy, however, 8 mM of TEMPOL was found to bemutagenic usingS. typhimuriumtester strain TA104, butthis was observed only in the presence of a superoxide-generating system; only minimal effects were observedwhen TEMPOL alone was used [41]. These conflictingresults with TEMPOL have not yet been fully inter-preted; nevertheless, in general, most studies concludethat there is no real evidence that the nitroxides investi-gated to date are themselves mutagenic [10,38]. Theresults reported in this present study demonstrate thataromatic indolinonic and quinolinic nitroxides are alsononmutagenic and lend support to the data in the litera-ture supporting the nonmutagenicity of nitroxide radi-cals.

The possible cytotoxicity of compounds1 through7was evaluated and compared with that of BHT usingCHO cells, a method employed in a number of studies onthe potential toxicity of nitroxides. In fact, the use ofassays of the ability of cells to form visible colonies(CFA), which requires that the cells successfully undergoseveral mitotic cycles, is considered to be one of the bestmeans to determine if test compounds will affect thelong-term status of the system. The results relative to thecytotoxicity of the compounds studied are reported inFig. 2, where the cytotoxic activity is expressed as the

percentage of relative survival (see Materials and Meth-ods). For all the compounds tested, with the exception of2 and5, a dose-dependent CFA inhibition was observed.In particular, for BHT and compounds6 and 7 at thehighest concentrations used (which corresponds to theirmaximum solubility), complete cell mortality was at-tained. At the highest concentrations tested for nitroxides1, 3, and 4, approximately a 50% decrease in survivalwas observed, whereas nitroxides2 and 5 induced nosignificant cytotoxic effects, as their relative percentagesof survival showed a mortality of about 10% at themaximum dose tested.

Because the cytotoxicity data express the activityexhibited by the compounds at different concentrations,to obtain a unitary parameter on the effective possiblecytotoxicity, the effective dose that inhibits the CFA by50% (IC50) (Table 2) was calculated. For compounds2and 5, the IC50 was not calculable, as at the highestconcentration (i.e., the highest concentration soluble inDMSO), the effects measured were less than 50%. Oncomparing the IC50 values with each other and above all

Fig. 2. Inhibition of growth of CHO/AS52 cell line treated for 5 h with compounds1 through7 and BHT. Each dose-response curvewas determined as described in Materials and Methods.

Table 2. IC50 Values Derived From the CFA Test inCHO/AS52 Cells Treated for 5 h With BHT and

Compounds1 Through7

Compound IC50 (mM)

BHT 576 4.51 746 6.62 —3 1226 3.14 416 3.25 —6 2416 5.87 116 1.2

— 5 not calculable.

334 E. DAMIANI et al.

with those of BHT, it can be observed that compounds1,3, and6 on an equal molar concentration basis are lesstoxic than BHT, although compounds4 and7 are 1.4 and5 times more toxic than BHT, respectively.

From this study, it seems that a structure-activityrelationship determined by the type of ring system and itssubstituents (to which the nitroxide function is attached)could exist. In fact, it seems that the aliphatic piperidinenitroxide TEMPO (5) is less toxic than the pyrrolinenitroxide TMPO (6) and that both are less toxic than thearomatic nitroxides. It has been previously reported thatpyrrolidinic nitroxides are more toxic than piperidinics,and this statement could also hold true for pyrrolinics[38]. In addition, it seems that the existence of thebenzene rings in the aromatic nitroxides could influenceand account for their cytotoxicity. This can be deducedfrom the fact that1 through3 (1 is more toxic than2,which, in turn, is more toxic than3) all correlate wellwith the number of aromatic rings present in their re-spective structures. One other point worth mentioning isthat the quinolinic nitroxide (4) is more cytotoxic thanthe indolinonic nitroxides (1–3). At present, the reasonfor this cytotoxicity remains to be clarified; however, apossible explanation for this could be the uncoupling ofenzymes, which causes the quinolinic nitroxide to shuntelectrons to itself and results in the production of reactivesuperoxide radical, as is the case with biologically activequinones like menadione [17].

In summary, the results are comforting, because allthe compounds tested except for4 and7 induced a levelof toxicity in mammalian cells cultured in vitro that isclearly lower (#8 times) than that exhibited by BHT, asynthetic antioxidant widely used in the food industry[42]. In addition, in this study, at the concentrations atwhich the nitroxides normally exert protection againstoxidative stress in the systems so far studied (10–50mM), no marked cytotoxicity is observed apart from thetwo exceptions mentioned above. The results presentedhere concerning the toxicity of these aromatic nitroxidesare in general agreement with those found in the litera-ture reporting on aliphatic nitroxides. The survival ofmonolayered CHO cells was unaffected by the piperidi-nic nitroxides TEMPOL and TEMPAMINE, the pyrro-linics CTPO and CTPC, and the lipid-soluble spin probes5-doxyl-, 12-doxyl-, and 16-doxylstearate at concentra-tions as high as 1 mM [39]. Similar results were obtainedwhen no effect of 10mM of TEMPOL on the survival ofcultured CHO AS52 cells could be detected [50] andwhen no effect of 10mM to 10 mM of TEMPO incubatedfor 2 to 48 h on the survival of MCF-7 human breastcancer cells could be detected [51].

Finally, although the results are encouraging and sup-port the increasing interest in nitroxides as potentialtherapeutic antioxidants, further in vivo and in vitro

toxicity studies are required before they can be actuallyconsidered for clinical trials. Furthermore, nitroxideshave recently been shown to possess a low potential tocause acute or subacute skin toxicity, and this mightsuggest their implication as possible novel antioxidantsfor cosmetic preparations [52].

Acknowledgements— This work was supported by an ex 60% grantfrom MURST (Ministero dell’Universita` e della Ricerca Scientifica eTecnologica). The authors also thank the University of Ancona forfinancial support.

REFERENCES

[1] Kocherginsky, N.; Swartz, H. M. Introduction. In: Kocherginsky,N.; Swartz, H. M., eds.Nitroxide spin labels, reactions in biologyand chemistry. Boca Raton: CRC Press; 1995:1–14.

[2] Rozantsev, E. G.Free nitroxyl radicals. New York: PlenumPress; 1970.

[3] Berliner, L. J.; Reuben, J.Spin labeling I and II, theory andapplications(Vols. 1 and 2). New York: Academic Press; 1976–1979.

[4] Hamilton, C. L.; McConnell, H. M. In: Rich, A; Davidson, N.,eds.Structural chemistry and molecular biology. San Francisco:W.A. Freeman; 1968:115–149.

[5] Swartz, H. M.; Feig, E.Advances in magnetic resonance imaging.Norwood, NJ: Ablex Publishing Co.; 1988.

[6] Berliner, L. J.; Fujii, H. Magnetic resonance imaging of biologicalspecimens by electron paramagnetic resonance of nitroxide spinlabels.Science227:517–519; 1985.

[7] Sosnovsky, G.; Li, S. W. In the search for new anticancer drugsXII. Synthesis and biological evaluation of spin labeled nitro-soureas.Life Sci. 36:1479–1483; 1985.

[8] Konovalova, N. P.; Diatchkovskaya, R. R.; Volkova, L. M.;Varfolomeev, V. N. Nitroxyl radicals decrease toxicity of cyto-static agents.Anticancer Drugs2:591–595; 1991.

[9] Krishna, M. C.; Samuni, A. Nitroxides as antioxidants.MethodsEnzymol. 234:580–589; 1994.

[10] Samuni, A.; Krishna, M. C. Antioxidant properties of nitroxidesand nitroxide SOD mimics. In: Packer, L.; Cadenas, E., eds.Handbook of synthetic antioxidants.New York: Marcel Dekker;1997:351–373.

[11] Mitchell, J. B.; Samuni, A.; Krishna, M. C.; De Graff, W. O.;Ahn, M. S.; Samuni, U.; Russo, A. Biologically active metal-independent superoxide dismutase mimics.Biochemistry29:2802–2807; 1990.

[12] Samuni, A.; Winkelsberg, D.; Pinson, A.; Hahn, S. M.; Mitchell,J. B.; Russo, A. Nitroxide stable radicals protect beating cardio-myocytes against oxidative damage.J. Clin. Invest. 87:1526–1530; 1991.

[13] De Graff, W. G.; Krishna, M. C.; Kaufman, D.; Mitchell, J. B.Nitroxide-mediated protection against X-ray- and neocarzinosta-tin-induced DNA damage.Free Radic. Biol. Med.13:479–487;1992.

[14] Miura, Y.; Utsumi, H.; Hamada, A. Antioxidant activity of ni-troxide radicals in lipid peroxidation of rat liver microsomes.Arch. Biochem. Biophys. 300:148–156; 1993.

[15] Samuni, A.; Godinger, D.; Aronovitch, J.; Russo, A.; Mitchell,J. B. Nitroxides block DNA scission and protect cells fromoxidative damage.Biochemistry30:555–561; 1991.

[16] Krishna, M. C.; Halevy, R. F.; Zhang, R.; Gutierrez, P. L.;Samuni, A. Modulation of streptonigrin cytotoxicity by nitroxideSOD mimics.Free Radic. Biol. Med. 17:379–388; 1994.

[17] Zhang, R.; Hirsh, O.; Mohsen, M.; Samuni, A. Effects of nitrox-ide stable radicals on juglone cytotoxicity.Arch. Biochem. Bio-phys. 312:385–391; 1994.

[18] Weil, T. J.; Van der Veen, J.; Olcott, H. S. Stable nitroxides aslipid antioxidants.Nature219:168–169; 1968.

335Genotoxicity and cytotoxicity of nitroxide radicals

[19] Bowry, V. W.; Ingold, K. U. Kinetics of nitroxide radical trap-ping. 2. Structural effects.J. Am. Chem. Soc. 114:4992–4997;1992.

[20] Nilsson, U. A.; Olsson, L. I.; Carlin, G.; Bylund, F. A. Inhibitionof lipid peroxidation by spin labels. Relationship between struc-ture and function.J. Biol. Chem. 264:11131–11135; 1989.

[21] Stipa, P.; Carloni, P.; Greci, L.; Damiani, E. Synthesis and ther-mal stability of alkoxyamines.Polymer Degradation and Stability55:323–327; 1997.

[22] Carloni, P.; Greci, L.; Stipa, P.; Eberson, L. Electron-transferreactions. Oxidation of Grignard reagents in the presence of anaminoxyl as a radical trapping agent.Journal of Organic Chem-istry 56:4733–4737; 1991.

[23] Damiani, E.; Carloni, P.; Stipa, P.; Greci, L. Reactivity of anindolinonic aminoxyl with superoxide anion and hydroxyl radi-cals.Free Radic. Res. 31:113–121; 1999.

[24] Greci, L. Homolytic substitution in indolinone nitroxide radicals.Part III. Reactions withterbutoxy and methyl radicals.Tetrahe-dron 38:2435–2439; 1982.

[25] Cardellini, L.; Carloni, P.; Greci, L.; Stipa, P.; Faucitano, A.Homolytic substitution in indolinone nitroxide radicals. Part 5.Reactions withtert-butylperoxy radicals.Gazzetta Chimica Itali-ana 119:621–625; 1989.

[26] Berti, C.; Colonna, M.; Greci, L.; Marchetti, L. Homolytic sub-stitutions in indolinone nitroxide radicals 1. Reaction with aroy-loxyl radicals.Tetrahedron33:3149–3154; 1977.

[27] Damiani, E.; Carloni, P.; Iacussi, M.; Stipa, P.; Greci, L. Reac-tivity of sulphur-centered radicals with indolinonic and quinolinicaminoxyls.European Journal of Organic Chemistry2405–2412;1999.

[28] Alberti, A.; Greci, L.; Stipa, P.; Sgarabotto, P.; Ugozzoli, F.Homolytic substitution in indolinone nitroxide. IV. Reaction withaminyl radicals. Spectroscopic and crystallographic study.Tetra-hedron43:3031–3040; 1987.

[29] Antosiewicz, J.; Bertoli, E.; Damiani, E.; Greci, L.; Popingis, J.;Przybylski, S.; Tanfani, F.; Wozniak, M. Indolinonic and quino-linic aminoxyls as protectants against oxidative stress.FreeRadic. Biol. Med. 15:203–208; 1993.

[30] Damiani, E.; Paganga, G.; Greci, L.; Rice-Evans, C. Inhibition ofcopper-mediated low density lipoprotein peroxidation by quino-line and indolinone nitroxide radicals.Biochem. Pharmacol. 48:1155–1161; 1994.

[31] Antosiewicz, J.; Popinigis, J.; Wozniak, M.; Damiani, E.; Carloni,P.; Greci, L. Effects of indolinic and quinolinic aminoxyls onprotein and lipid peroxidation of rat liver microsomes.FreeRadic. Biol. Med. 18:913–917; 1995.

[32] Antosiewicz, J.; Damiani, E.; Jassem, W.; Wozniak, M.; Orena,M.; Greci, L. Influence of structure on the antioxidant activity ofindolinic nitroxide radicals.Free Radic. Biol. Med. 22:249–255;1997.

[33] Tanfani, F.; Carloni, P.; Damiani, E.; Greci, L.; Wozniak, M.;Kulawiak, D.; Jankowski, K.; Kaczor, J.; Matuskiewicz, A.Quinolinic aminoxyl protects albumin against peroxyl radicalmediated damage.Free Radic. Res.21:309–315; 1994.

[34] Villarini, M.; Moretti, M.; Damiani, E.; Greci, L.; Santroni,A. M.; Fedeli, D.; Falcioni, G. Detection of DNA damage instressed trout nucleated erythrocytes using the comet assay: pro-tection by nitroxide radicals.Free Radic. Biol. Med. 24:1310–1315; 1998.

[35] Damiani, E.; Greci, L.; Parsons, R.; Knowland, J. Nitroxideradicals protect DNA from damage when illuminated in vitro inthe presence of dibenzoylmethane and a common sunscreen in-gredient.Free Radic. Biol. Med. 26:809–816; 1999.

[36] Gabbianelli, R.; Falcioni, G.; Santroni, A.; Caulini, G.; Greci, L.;Damiani, E. Effect of aromatic nitroxides on hemolysis of humanerythrocytes entrapped with isolated hemoglobin chains.FreeRadic. Biol. Med. 23:278–284; 1997.

[37] Falcioni, G.; Gabbianelli, R.; Damiani, E.; Santroni, A. M.;Fedeli, D.; Wozniak, M.; Greci, L. The effect of indolinic andquinolinic nitroxide radicals on trout erythrocytes exposed tooxidative stress.Free Radic. Res. 28:507–516; 1998.

[38] Kocherginsky, N.; Swartz, H. M. Sentjurc, M. Toxicity and theuse of nitroxides as drugs. In: Kocherginsky, N.; Swartz, H. M.,eds.Nitroxide spin labels, reactions in biology and chemistry.Boca Raton: CRC Press; 1995:175–198.

[39] Ankel, E. G.; Lai, C.-S.; Hopwood, L. E.; Zivkovic, Z. Cytotox-icity of commonly used nitroxide radical spin probes.Life Sci.40:495–498; 1987.

[40] Sosnovsky, G. A critical evaluation of the present status oftoxicity of aminoxyl radicals.J. Pharm. Sci. 81:496–499; 1992.

[41] Sies, H.; Mehlhorn, R. Mutagenicity of nitroxide-free radicals.Arch. Biochem. Biophys.251:393–396; 1986.

[42] Aruoma, O. I.; Halliwell, B.Free radicals and food additives.London: Taylor and Francis; 1991.

[43] Colonna, M.; Greci, L.; Berti, C.; Marchetti, L. Stable nitroxideradicals from phenyl-isatogen and arylimino derivatives with or-ganometallic compounds.Tetrahedron31:1745–1753; 1975.

[44] Berti, C.; Colonna, M.; Greci, L.; Marchetti, L. Nitroxide radicalsfrom 2-substituted quinoline-N-oxides with organometallic com-pounds.Tetrahedron32:2147–2151; 1976.

[45] Rozantsev, E. G.; Krinitskaya, L. A. Free iminoxyl radicals in thehydrogenated pyrrole series.Tetrahedron21:491–499; 1965.

[46] Maron, D. M.; Ames, B. N. Revised methods for the Salmonellamutagenicity test.Mutat. Res. 113:173–215; 1983.

[47] Hrelia, P.; Vigagni, F.; Maffei, F.; Fimognari, C.; Lamartina, L.;Spinelli, D.; Juric, P.; Guerra, M. C.; Cantelli-Forti, G. Analysisof metabolism and genotoxicity of 5-nitro-3-thiophenecarboxani-lides in bacterial, mammalian and human cells.Mutagenesis10:171–177; 1995.

[48] Afzal, V.; Brasch, R. C.; Nitecki, D. E.; Wolff, S. Nitroxyl spinlabel contrast enhancers for magnetic resonance imaging. Studiesof acute toxicity and mutagenesis.Invest. Radiol. 19:549–554;1984.

[49] Johnstone, P. A.; De Graff, W. G.; Mitchell, J. B. Protection fromradiation-induced chromosomal aberrations by the nitroxideTEMPOL. Cancer75:2323–2327; 1995.

[50] De Graff, W. G.; Krishna, M. G.; Russo, A.; Mitchell, J. B.Antimutagenicity of a low molecular weight superoxide dis-mutase mimic against oxidative mutagens.Environ. Mol. Muta-gen.19:21–26; 1992.

[51] Voest, E. E.; Van, R. E.; Van, A. B.; Neijt, J. P.; Marx, J. J.Increased hydrogen peroxide concentration in human tumor cellsdue to a nitroxide free radical.Biochim. Biophys. Acta1136:113–118; 1992.

[52] Fuchs, J.; Groth, N.; Herrling, T. Cutaneous tolerance to nitroxidefree radicals in human skin.Free Radic. Biol. Med. 24:643–648;1998.

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