Aqueous Extracts of Cigarette Tar Containingthe Tar Free Radical Cause DNA Nicks inMammalian CellsKoni K. Stone, Eliezer Bermudez, and William A. PryorBiodynamics Institute, Louisiana State University, Baton Rouge, Louisiana

The ability of aqueous extracts of cigarette tar to nick DNA was investigated using viable mammalian cells. Tar extracts contain a radical with a stableelectron spin resonance (ESR) signal at g = 2.0036 characteristic of a semiquinone. The association of the tar component that carries the ESR signalwith DNA was demonstrated using viable rat alveolar macrophages. The formation of single-strand DNA breaks caused by cigarette tar extracts inviable rat thymocytes follows saturation kinetics, indicating a tar component associates with DNA and then nicks it. These studies support ourhypothesis that tar components that contain the cigarette tar radical can enter cells, associate with, and then nick DNA. - Environ Health Perspect102(Suppl 10):173-178 (1994)

Key words: semiquinone radicals, autoxidation, hydrogen peroxide, catalase, superoxide dismutase, glutathione, cancer

IntroductionCigarette smoking is a major cause of

human lung cancer (1), and the evidencesuggests that radical mechanisms are

involved in causing the DNA damage thatcould lead to cancer (2,3). Cigarette tar

contains high concentrations (>1017spins/gram) of stable radicals that can beobserved directly by electron spin reso-

nance (ESR) (4). At least four radicals havebeen identified based on their ESR spectralcharacteristics; the most interesting is a

semiquinone in equilibrium with quinonesand hydroquinones in a low molecularweight, tar matrix (2,4,5).

For each commercial cigarette smoked,up to 12 mg of particulate material(excluding nicotine and water) is depositedin the lungs of a smoker (6). This materialcomes in contact with pulmonary fluidsthat wash over it and extract the water-sol-uble components. Therefore, we believeaqueous cigarette tar extract (ACT) solu-tions are a realistic model of the mixture ofchemicals that lung cells are continuouslyexposed to in the lungs of a smoker.

The tar fraction that contains thesemiquinone radical associates with DNA(7). When calf thymus DNA is incubated

This paper was presented at the Conference onOxygen Radicals and Lung Injury held 30 August-2September 1993 in Morgantown, West Virginia.

This work was supported by National Institutes ofHealth grant HL 25820. We are grateful to H. C.Birnboim for helpful discussions.

Address correspondence to Willaim A. Pryor,Biodynamics Institute, Louisiana State University,711 Choppin, Baton Rouge, LA 70803-1800.Telephone (504) 388-2063. Fax (504) 388-4936.

with buffer solutions containing the tarradical, the DNA recovered by precipita-tion contains the tar radical ESR signal,and this signal is not removed by washingthe DNA (7). Aqueous cigarette tarextracts nick plasmid DNA (8), producingnicks that are not easily repairable (9);thus, error-prone mechanisms may beinvolved in the repair of the tar radical-induced nicks and these erroneous repairscould lead to mutations. When plasmidDNA is incubated with ACT, the resultantDNA nicking appears to follow saturationkinetics (10). Based on these preliminaryDNA nicking studies, we have proposed amodel in which the tar radical first associ-ates with, and then nicks DNA (3).

It is our hypothesis (2) that superoxideand hydrogen peroxide (H202) result fromthe reduction of dioxygen by the hydro-quinones and/or semiquinone radicals pre-sent in smoke extracts (Equations 1-3).

QH2+O2 - QH-+0j+H+ [1]

QH++02 - Q+O-+H+ [2]

20- + 2H > H202 + 02 [3]

H202 can then be reduced to the hydroxyl(HO-) radical by metals such as iron(Equation 4), and the resulting HO- radi-cal can then nick DNA (3,11).

H202 + Fe+2 - HO + OH- + Fe+3 [4]

The iron in reaction 4 probably is com-plexed to the DNA via polyhydroxyaro-

matic components in the tar (3,11). Infact, Ghio et al. have found that aqueouscigarette tar extracts complex iron in vitroand in vivo (personal communication).We report here studies that show the

tar radical component in ACT producesDNA nicks using viable mammalian cells.We also report, for the first time, ESRspectra of the tar radical in aqueous solu-tions and show that the number of DNAnicks produced is proportional to theamount of tar present. We also report theeffects of reduced glutathione (GSH),deferoxamine, catalase, and superoxide dis-mutase (SOD) on the yield of DNA nickscaused by tar extracts.

Materials and MethodsMaterialsChemicals and enzymes (unless notedotherwise) were purchased from SigmaChemical Co. (St. Louis, MO) and usedwithout further purification. Reducedglutathione, 800 mM, pH 8.1, catalase 1mg/ml, and SOD 1 mg/ml were all pre-pared just before use. Catalase was inacti-vated by boiling for 2 min in 1 N NaOHand then the pH was adjusted to 7.4 withHCl; SOD was inactivated by boiling for 2min. Inactivated enzyme solutions werestored at 40C.

Preparation ofACTResearch grade cigarettes (1R2) from theKentucky Tobacco Research Council weresmoked to a butt length of 30 mm using astandard puff profile (30 ml puff/30 sec).The tar from four cigarettes was collected

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STONE ETAL.

on a Cambridge filter (a glass fiber filterthat retains 99.9% of the particles largerthan 0.1 micron), and the filter wasextracted with 5 ml of pH 8 PBS (phos-phate-buffered saline: 0.066 M phosphate,0.85%, w/v, NaCl). After standing in thedark at 370C for 24 hr, the solution was fil-tered with a 0.2-pm filter (25 mm,polypropylene, Whatman InternationalLtd., Mainstone, England). This methodproduces ACT solutions that contain 8 to10 mg of tar/ml of solution. An alternatemethod was employed to increase the taryield. Tar deposits were washed off aCambridge filter with approximately 15 mlof acetone until the filter was colorless, andthe acetone was removed by evaporationwith a stream of nitrogen. The driedresidue was then resuspended in 5 ml ofPBS, pH 8, in a vial and the solution wassonicated for 15 min using a Branson 2200sonicator (Branson Ultrasonics). Afterstanding at 37°C in the dark for 24 hr, thisACT solution was filtered with a 0.2-pmfilter (25 mm, polypropylene, Whatman).This second method yields ACT solutionsthat contain 20 to 27 mg of tar/ml. Webelieve this second method ofACT prepa-ration yields the same kind of tar only in ahigher concentration. Therefore, for eachexperiment the volume of ACT solutionused was adjusted to keep the amount oftar constant.

Separation ofACT byMembrane FiltrationUsing Centricell 30 K and 10 K membranefilters (Polyscience, Inc., Warrington, PA),three molecular weight (mw) fractions oftar were obtained: fraction 1 >30,000 mw,fraction 2 between 10,000 and 30,000 mw,and fraction 3 < 10,000 mw. The amountof tar was determined to be 1.0 mg/ml, 1.6mg/ml, and 24.4 mg/ml for fractions 1, 2,and 3, respectively. For each experiment,the volume of each fraction used wasadjusted to keep the concentration of tarconstant.

Isolation ofAlveolar Macrophagesand ThymocytesA male Sprague-Dawley specific pathogen-free rat 92 to 94 days old (360-400 g)(Harlan Sprague-Dawley, Houston, TX)was anesthetized with phenobarbital andthen sacrificed by exsanguination. The thy-mus was removed, placed in a normalsaline solution and then mashed withtweezers to release the thymocytes. Thecells were pelleted by centrifugation at400g and resuspended in 0.87% ammo-nium chloride/10 mM Tris-HCl, pH 7.2,

10 mM sodium bicarbonate solution tolyse any red blood cells. After 20 min, thecells were centrifuged at 400g and resus-pended in a buffered isotonic solution, pH7.2, containing 0.25 M myoinositol, 10mM sodium phosphate, and 1 mM magne-sium chloride. The thymocytes werecounted, and their viability determinedusing the trypan blue exclusion assay. Thisprocedure yields 1.5 to 1.8 x 109 cells with90 to 95% viability.

To harvest macrophages from lung, 10ml of a normal saline solution (37°C) wasinstilled intratracheally into the lungs, thesolution was allowed to remain for oneminute, and then recovered. This wasrepeated three times and the lavage solu-tions were pooled. Alveolar macrophageswere separated from the lavage solutions bycentrifugation at 400g. Typically, 5 to 6million cells with 85 to 90% viability wereharvested.

DNA Nlcking in ThymocytesThymocytes (17 million cells/tube, 1.2 mltotal volume) were incubated on ice withaliquots of ACT for 90 min. For experi-ments involving inhibitors, the inhibitorwas added to the cells prior to the incuba-tion. The cells were then pelleted by cen-trifugation and resuspended in a bufferedisotonic solution, pH 7.2, containing 0.25M myoinositol, 10 mM sodium phosphate,and 1 mM magnesium chloride. The fluo-rescence analysis of DNA unwinding(FADU) assay has been described in detailby Birnboim (12,13). Briefly, the cells aredivided into three types of tubes: T (totaldouble-stranded DNA) tubes, in whichcare is taken to prevent DNA unwinding,indicate the total (maximum) amount ofdouble-stranded DNA present. P (partiallyunwound DNA) tubes, which are treatedwith a viscous alkaline solution that allowsfor partial unwinding of the DNA, are usedto assess DNA damage. B (blank, no dou-ble-stranded DNA) tubes, are treated withthe viscous alkaline solution and then soni-cated with a Branson sonifier 450, 15pulses at 20% output (Branson Ultra-sonics) to effect maximum unwinding ofthe DNA, and contain no double-strandedDNA. At this stage, the volume of eachtube is 1.0 ml, then 1.5 ml of a 6.67 pg/mlsolution of ethidium bromide is added toeach tube. The amount of double-strandedDNA in each tube is determined by mea-suring the fluorescence of ethidium bro-mide using 520 nm excitation and 590 nmemission. Three of each type of tubes, T, P,and B, are used for each determination,and the results are averaged. The amount

of double-stranded DNA remaining is cal-culated from Equation 5:

D = [(P-B)/(T-B)] x 100% [5]

The amount DNA damage is determinedby calculation of the damage quotient Qd,as shown in Equation 6:

Qd = (log DC-log DACT) X 100 [6]

where DC = double-stranded DNA in con-trol cells and DA,T = double-stranded DNAin ACT-treated cells (12). Using these cal-culations, Birnboim demonstrated a linearrelationship between Qd and the amountof cobalt-60 gamma radiation that lympho-cytes received (12). The amount of protec-tion provided by the various inhibitors iscalculated as shown in Equation 7:

% Pro- = (1 Qdinhibitor/QdAC) x 100tection [7]

Binding of the Cigaette Tar Radical toDNA in Rat Alveolar Marophages

RAM, isolated as described above, wereresuspended in 2.0 ml of a solution con-taining 24 mM EDTA and 75 mM NaCl(the solution was adjusted to pH 7.5 with1 N NaOH). The cells were then incu-bated with 1.0 ml of an ACT solution, or1.0 ml of PBS, pH 8.0. In both incuba-tions, the cells were allowed to sit on ice for90 min to allow for maximum penetrationof the tar into viable cells. The cell viabilitydecreased markedly for incubations longerthan 90 min. After the incubation, exclu-sion of trypan blue dye was used to deter-mine cell viability to be 85 to 90%.Modification of the alkaline elution proce-dure (14) was used to isolate double-stranded DNA on polycarbonate (pc)filters. A Micro/Por Polycarbonate mem-brane (25 mm, 0.2 pm, Spectrum MedicalIndustries, Inc., Los Angeles, CA) wassoaked in ice-cold PBS, pH 7.4, for 20min, and then placed in an alkaline elutionfunnel (Millipore, Bedford, MA). A smallvolume of PBS was run through the filterto check for leaks and then RAM in PBSwere carefully deposited on the filter usinga slow flow rate. A peristaltic pump is usedto pull fluid through the filter at flow of 1ml/min. After loading the cells on to thefilter, 10 ml of a lysis solution (2% SDS,0.025 M EDTA, pH 9.7) is added andallowed to remain in the funnel above thefilter at room temperature for 60 min. Thissolution is then pulled through the filterand 4 ml of the lysis solution with added

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DNA DAMAGE BY CIGARETTE TAR

proteinase K (0.5 mg/ml) is poured intothe funnel and the cells are allowed toincubate at room temperature for an addi-tional 30 min. The proteinase K solution ispulled through the filter and the filterwashed with 10 ml of PBS and then airdried. These steps lyse the cells and removeprotein, RNA, and any single-strandedDNA. The filter retains large pieces ofdouble-stranded DNA. Control experi-ments included incubations of cells withno ACT, and incubations with ACT andno cells. The dried filters were shreddedwith scissors and packed into quartz tubesfor examination by ESR spectroscopy.

Electron Spin Resonance SpectroscopyESR spectra of aqueous solutions weredetermined in a quartz flat cell using aVarian E-109 X-band spectrometeremploying 100-kHz modulation. Radicalconcentrations were estimated by doubleintegration and g values were determinedusing Fremy's salt (potassium nitroso disul-fonate) as a standard for both determina-tions (15).

For dried samples and organic solu-tions, spectra were measured using aBruker ER IOOD X-band spectrometerwith 100-Khz modulation frequency. Theg values were determined by comparisonwith DPPH (15).

Resultsracton ofthe Tar Radical into

Aqueous Buffer SolutionsPreviously, we have reported ESR spectra ofthe tar radical in organic solvents or on a fil-ter (4). The tar radical signal is extractedinto aqueous buffers at pH 8.0 and its ESRspectrum observed in aqueous buffers(Figure 1). These ACT solutions contain abroad ESR signal with a g value of 2.0035,which we have assigned to the tar semi-quinone radical (4). This g value is typical oforganic semiquinones (16) and organic semi-quinone radicals previously have beenobserved in aqueous solutions at pH 8.0 (17).

DNA-Tar Radical Complexes Formedwith Caf-Thymus DNATar extracts contain stable semiquinoneradicals that become associated with dou-ble-stranded DNA (7). In preliminaryexperiments, calf thymus DNA was incu-bated with ACT for 18 hr at 37°C and theDNA precipitated with cold ethanol. TheESR spectra of the ethanol precipitatesfrom incubation of ACT with thymusDNA are shown in Figure 2. The treatmentof ACT alone with cold ethanol does not

result in the precipitation of any material.The DNA that precipitated from ACT-DNA incubations had an ESR signal with ag value of 2.0044, in agreement with ourprevious report (7) and within the rangefor semiquinone radical signals (16).

Next, calf thymus DNA was incubatedwith ACT and the DNA was isolated on apolycarbonate filter that impedes the pas-sage of long DNA strands (14). Theseincubations result in the semiquinone radi-cal becoming retained by the filter; Figure3 shows the ESR spectrum observed fromthe dried polycarbonate filter. Controlincubations of ACT alone, or calf thymusDNA alone, showed no radical signalremaining on the filter. This indicates thatthe DNA-tar radical complex is retainedon the polycarbonate filter, and there is nononspecific retention of the tar radical onthe filters in the absence ofDNA.

Ig= 2.0047

A

B

1 0 gauss

Figure 1. Comparison of the ESR spectra of the tarradical in t-butyl benzene (A) and the tar radicalextracted into aqueous phosphate buffer at pH 8.0 (B).

-. 2.0044A

DNA-Tar Radical Complex inRat Alveolar MacrophagesSince the DNA-radical complex can betrapped on a polycarbonate filter, incuba-tions of ACT with viable RAM cells wereperformed. RAM were incubated withACT solutions and the DNA was immobi-lized on polycarbonate filters using amodification of the alkaline elutionmethod of Kohn et al. (14). The ESRspectra of the dried filters are shown inFigure 4. The ACT solution contains aradical that associates with the double-stranded DNA in RAM, and the dried fil-ters show this ESR spectrum. The controlincubation of RAM cells alone or ACTalone show no ESR signal.

g = 2.0039A

B

C

10 gauss

Figure 3. ESR spectra of material remaining on poly-carbonate filters after the following incubations: (A)ACT + calf thymus DNA; (B) ACT alone; (C) calf thymusDNA alone.

g - 2.0034g - 2.0005

A

g 2.0039 B

C

g.=2.0047 BD

1 0 gauss

Figure 2. Comparison of ESR spectra of dried ACT andACT co-precipitated with calf thymus DNA. (A)Ethanol precipitates of calf thymus DNA exposed toACT, after washing off the unbound ACT. (B) DriedACT alone.

Figure 4. ESR spectra of the material bound to poly-carbonate filters after the following incubations: (A)viable RAM + ACT <10,000 mw; (B) viable RAM +ACT; (C) viable RAM alone; (D) ACT, no cells. Detailsof the isolation and incubations of RAM are given inMaterials and Methods.

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STONE ETAL.

DNA Nlcking byAqueous racts ofCigarette TarThe results of the FADU assay for ACT-induced DNA nicks in rat thymocytes areshown in Figure 5. The DNA damage quo-tient (Qd) represents the amount of DNAnicks in the rat thymocytes. (The calcula-tion of Qd is explained in Materials andMethods.) When Qd is plotted versus thetotal concentration of tar in the aqueousextracts, the maximum Qd and the bindingconstant can be determined either from acurve-fitting program or by Lineweaver-Burk or Eadie-Hofstee plots of the data.These three analyses are summarized inTable 1, and the Lineweaver-Burk plot isshown in the insert of Figure 5. The maxi-mum value for Qd is 98 ± 13 and the bind-ing constant is 2144 ± 237 )Jg/ml for tarextracts. These data indicate saturationkinetics, with a maximum amount ofDNAdamage occurring at a tar concentration of2144 pg/ml. This is the amount of tar pro-duced by approximately 0.23 cigarette.

Membrane filters were used to separatethe ACT solution into three fractions, andthese fractions were tested for DNA nickingactivity. Only fraction number 3, the frac-tion containing material with MW less than10,000 amu, caused DNA nicks, as shownin Figure 6. This fraction also contains thesemiquinone radical that associates with theDNA in RAM, as shown in Figure 4.

Eflects ofInhibitors on DNA DamageCaused byACI SolutionsCatalase, SOD, GSH, deferoxamine, andmannitol were tested to determine if theyprotect DNA against nicking caused by tarextracts; the results are summarized inTable 2. Catalase and GSH protected thecells from DNA damage caused by ACTsolutions. As the GSH concentration wasincreased, the amount of protection againstDNA nicking by ACT also increased, asshown in Figure 7. Neither deferoxaminenor SOD protected against DNA nickingcaused by ACT solutions. These results aresummarized in Table 2.

DiscussionStudies in our laboratory with cigarette tar-induced DNA nicking were done with iso-lated plasmid DNA (8) or calf thymusDNA (7). We have extended our studies todetermine the effects of cigarette tar inviable cells. We have also shown that the tarradical can be extracted into aqueous buffersolutions and directly observed by ESR.[We had previously reported ESR spin trapspectra of the hydroxyl radical that is pro-duced by these ACT solutions (18).]

120

§ 00

a 40

ol

f ~~~~~~~~~~

0 2000 4000 6000ACT(p/ml

Figure 5. Plot of Qd (DNA damage quotient in rat thy-mocytes) versus the concentration of tar in ACT solu-tions. The equation for the calculation of Qd is given inMaterials and Methods. The insert shows theLineweaver Burk plot of these data.

Table 1. Comparison of data analysis for DNA nickingcaused by ACT solutions.

Maximum BindingMethod of Qd constantadata analysis (A) (B)

Curve-fitting (Figure 6) 112 1968f(Qdi) = A[ACTI/(B+[ACT])

Lineweaver-Burk plot 102 2480(insert of Figure 5)f(1/Qdi)= (B/A)(1/[ACT])+1/A

Eadie-Hofstee plot 80 1986f(Qdi) = -B(Qdi/[ACTJ)+A

Average 98 ± 13 2144 ± 237

aApparent binding constant units are microgram permilligram.

For DNA binding studieused RAM. These cells are targrette smoke damage (19-27) aiisolated as a homogeneous cell(28) from the lavage of rat lungmodified the alkaline elutionKohn et al. (14) so that itretains only double-strandedRAM on polycarbonate filters.method, we are able to isolateintact cells that have been expoand show that the tar radical lDNA in these cells.

For DNA nicking studiemployed the FADU method

80

X 40

20

0 50 100GSH (mM)

Figure 7. Plot of percent protectioninduced DNA nicks versus the concentraglutathione.

50

40-

30 -

820-

0-0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

Tar (ff / ml)

Figure 6. Plot of Qd (DNA damage quotient in rat thy-mocytes) versus the concentration of tar in the frac-tions of ACT solutions. Fraction 1 (O) is >30,000 mw;fraction 2 (*) is between 10,000 and 30,000 mw; andfraction 3 (o) is < 10,000 mw.

Table 2. Comparison of the effects of inhibitors forprotection against DNA nicking by ACT solutions.

% Protectionfrom ACT nicking, Number of

Inhibitor average ± SD experiments

Catalase 83.4 ± 12.1 6Boiled catalase 22.3 ± 20.0 3SOD 37.8 2Boiled SOD 34.7 2Catalase + SOD 97.8 2Deferoxamine, 0.1 mM 20.7 8.6 4Deferoxamine, 0.5 mM 28.1 ± 12.8 4Deferoxamine, 1.0 mM 26.89 + 9.2 4GSH, 200 mM 85.1 ± 7.1 3

(12). This method allows the detection ofDNA single-strand breaks in cells that have

s, we have been exposed to ACT. For these studies we;ets for ciga- have used rat thymocytes; while these arend are easily not primary target cells for DNA damagepopulation in smokers, large numbers are easily

gs. We have obtained as a homogeneous cell popula-method of tion. We are able to routinely isolate 1.5 toselectively 1.8 billion cells, the number required toDNA from complete a dose response curve. Therefore,Using this thymocytes are a useful and experimentallyDNA from accessible model system that we have used)sed to ACT in our initial probing of the effect ofACTbinds to the in viable cells (13).

Leanderson and Tagesson studied thees we have ability of aqueous cigarette tar extracts toof Birnboim promote human polymorphonuclear leuco-

cyte and hydrogen peroxide-induced DNAsingle-strand breaks in cultured human

+ bronchiolar cells (29). The tar alone didnot cause any single-strand DNA breaks.These workers used tar solutions that were8-fold more dilute than the most dilutestudied here, and apparently were toodilute to cause nicking. They also preparedtheir tar solutions differently than we, bub-

150 200 bling whole smoke through chloroform,evaporating the chloroform and redissolv-

against ACT- ing the tar in 10 ml of buffer.3tion of reduced Nakayama et al. reported that cigarette

smoke produces single-strand breaks in cul-

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DNA DAMAGE BY CIGARETTE TAR

tured human A549 lung carcinoma cellsand the number of breaks was reduced byradical scavengers (30). In their experi-ments, smoke extracts were prepared bybubbling the smoke from two commercialfiltered cigarettes into a phosphate buffer.The authors noted the large variability (5-fold) in their results when different solu-tions of smoke were employed, and theyattribute these difference to fluctuatingconditions in trapping the smoke and thevariable quality of commercial filters andcigarettes. Fielding et al. sought to deter-mine if the tar fraction of smoke extractscaused these single-strand breaks (31).These workers used the smoke from fourtypes of commercial cigarettes: ultra low,low, and medium tar with filters and hightar without a filter. The smoke was bub-bled through buffer and the extracts thentested for DNA nicking in cultured A549cells. These workers find no correlationbetween the amount of tar from a particu-lar cigarette and the extent of DNA nick-ing observed, and they conclude that tar isnot involved in DNA nicking. Our resultsindicating that aqueous tar extracts do nickDNA conflict with this conclusion.Neither Nakayama et al. nor Fielding et al.separated gas-phase cigarette smoke and tarby the standard methods we used, andboth used commercial cigarettes, whereaswe used standard research cigarettes.

We find that increasing amounts of tarextracts result in higher amounts of DNAdamage up to a maximum (saturation)point. Using DNA isolated from rat lungand a 32P-postlabeling assay for DNAadducts, Randerath et al. also found a cor-relation between the amount of DNAadducts and the amount of cigarette smokeextract used in the incubations (32). Theirresults, like ours, show that the yield ofadducts reaches a saturation maximum(32). These workers found that glutathioneinhibits adduct formation, and they pro-posed that radical mechanisms are involvedin the formation oftar-DNA adducts.

3. 02- is formed from QH2autoxidation and dismutates

H + to give H202 (which can2. Tar 02 H also diffuse in from other

semiquinones (Fe) cellular sites)reduce 02 e° 2 '-I H202to give 02' 4. Metals chelated

OH H2 )toDNA -bound

QH2/0OQH&species convert

1 A low molecular Tar speis 1H202 to HOweight polyhydroxy containing the HO* (or ferryl species)aromatic component Q/QH2/QH at nickDNAin tar binds to DNA complex t n DNA

, ~~~~~II

90ADNA

Scheme 1. Model of the tar radical associating with and nicking DNA. 1) Aqueous extracts of cigarette tar con-tain a semiquinone/hydroquinone radical in a low molecular weight polymeric matrix that binds to DNA. 2) Thesesemiquinones reduce oxygen to form superoxide. 3) Superoxide then dismutates to form hydrogen peroxide; orhydrogen peroxide may diffuse into the nucleus from other loci in the cell. 4) Metals chelated to the tar react withhydrogen peroxide to form hydroxyl radical (or ferryl) species that nick DNA. This figure has been modified fromPryor and Stone ( 1 1).

Cigarette smoke solutions have beenshown to produce hydrogen peroxide (33).The DNA nicking caused by ACT isinhibited by catalase; hydrogen peroxide isimplicated in causing DNA nicks in mam-malian cells exposed to ACT. Similarly,Evans et al. showed that catalase protectedagainst cigarette tar damage to the proteinalpha-1-proteinase inhibitor (34).

Glutathione protects against DNAnicking by ACT. Glutathione is known toform covalent adducts with quinones andhydroquinones (35-37). Thus, the GSHprotection of DNA may be related to theability of GSH to add to quinones andhydroquinones and perhaps prevent theaddition to and nicking of DNA, or to thewell known ability ofGSH to quench radi-cal signals (38).

Aqueous extracts of cigarette tar con-

tain a tar semiquinone radical. Theseextracts cause DNA nicking in intact cellsand this nicking follows saturation kinetics,suggesting that cigarette tar associates withDNA and then causes nicks (11). Themaximum nicking occurs at concentrationsof tar that are equivalent to 0.23 cigarette,using incubations of 17 x 106 thymocytes.The tar radical also associates with DNA inviable rat lung macrophages. These resultsshow that tar components are capable ofentering a viable cell, penetrating thenucleus, and interacting with DNA, andsupport our model of the tar radical associ-ating with, and then nicking, DNA (11) asshown in Scheme 1. This provides furtherevidence that the cigarette tar componentcontaining the free radical is involved inthe DNA damage and subsequent carcino-genicity of cigarette tar.

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