Document pBXbK2ev3NEQrrKGk9xpwObbd

OS/30/02 11:04 FAX 817 705 2283 UNT HEALTH SCIENCE @003 ARCyJVIS or BWCHBMUTRV AMD BIOPHYSICS VoL OIL No. L My is, pp. 18-18,1994 notice LrSr*WOo(iaH PLAINTIFF'S EXHIBIT DMA Strand Breaks Following in Vitro Exposure to Asbestos Increase with Surface-Complexed [Fe3+] Andrew J. Ghio,*'1 Thomas P. Kennedy,* Jacqueline G. Stonehuerner.t Alvin L. CrumbUss,t and John R. Hoidali *pivision of Allergy, Critical Care, and Respiratory Medicine, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710; 1Department af Chemistry, Duke Unwereity; Durham, North Carolina 27710; and %Respiratvry, Critical Care, and Occupational (Pulmonary) Medicine, Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84134 Receivad July 19,1903, and in nvited form February '41994 Surface functional groups on silicate dusts complex iron cations which can cycle through reduction and ox idation states to generate free radicals. Tbsse oxidants have a capacity to produce DNA strand breaks and mu tations which are primary events in cancer induction. A differential in the capacity of fibrous silicatss to produce carcinoma is recognised with the amphiboles demon strating a greater biologic effect than theserpentine fiber chrysotile. We tested tbe hypothesis that the differences in genotoxicity of these fibrous silicates correspond to varying concentrations of iron complaxed to the surface. Relative to chrysotile, the amphibolo fibers complexod greater amounts of iron cations from both inorganic and tn vivo sources. Increased concentrations of surfacecomplexed iron were associated with greater oxidant generation, measured as tbiobarbituric add-reactive product* of deoxyriboee, and more covalently dosed, circular DNA strand scission. These results indicate that geuotoxic effects of these fibers may correspond to their capacity to complex iron at the surface. * um tatw, PllW.hc. genotoxicity and cancer induction suggests that oxidants generated by fibrous silicates are associated with DNA damage (3,4). Acidic functional groups on the surface of mineral oxides have a capacity to complex transition metals, especially iron, which catalyze electron transport (8), Free radicals are generated during the oxidation of ferrous to ferric ions; Fe*+ + H*0.> ^ Fo3+ + *OH + 'OH ... Fe2+ + H,0, ss Fc(OH)i+. Tbe products of the reaction between Fe** and HjOa are debated, but both hydroxyl COH) and ferryl (Fe(OH)J+) radicals can affect DNA strandbreaks (6,7), In addition, oxidants could possibly function to activate oncogenes or inactive anti-oncogenes (8, 9). A differential in the ca pacity, of fibrous silicates to produce mesothelioma and carcinoma is recognized with the amphiboles, includ ing Kmosite (iFe^.MgMSiiOaHOHji) and crocldolite Inhalation of fibrous silicates increases the incidence of lung neoplasms and mesothelioma. The mechanism of cancer induction is not known. Carcinogenicity ia pos tulated to reflect parameters ofparticle geometry and tbe contingent capacity of the lung to clear the dust (1). In (Na1(Fa*'t']j[Fe3+]i[Si#0*iKOH]i), demonstrating a bio logic effect greater than that of the serpentine fiber chiysotile <Mg3[Si*05] [OH]4) (10,11). We tested tire hypoth: eais that the differences in genotoxicity of silicates correspond to varying concentrations of iron complexed to the fiber surface which can catalyze the generation of oxidants and therefore affect DNA damage. * ability to move the longer, thinner fibers from the lung is proposed to result in their endocytosis, potential in MATERIALS AND METHODS ' teraction with the mitotic apparatus oftbe cell, and chro mosomal aberrations (2). An alternative hypothesis of Materials. AU material* ward obtained (ratb. Sigma Co. (3l LooJk, MO) unlm specified. Buffer* ware treated with cbalatinc min (Chafe* 10 r/BOO ml) for 2 h with agitation to diminish aveiUbb iron cation*. , * To whom corrcrpondanc* should h addraaaed at Dak* Uctirasiiy ClvssaUrnrasian ofsilicates- Th* aurfaoe araaa of amoalta, crocldolite, Madicftl Cantor, Box 3177, Durham, NC 27710. and ciuyeotile (Naffnnul Institute of Environmental Health Sciences, . M03-9SS1/VH $6.00 Copyrigfal < 1994 by Acdnsio Piw, Inc: All ri*hu of reproduction in any form reserved. . 13 I RECEIVED FIMEmJUM. 20. || |: 32AS [PRINT T1MEBWUN. 20. "11:42AM 06/30/02 11:34 FAX 817 735 2283 (JNT HEALfH SCIENCE @004 9 14 OHIO BT AL, Rerearch Triangle Pork, NC) wsr# determined using tbs Brunur-Km- meto-Tell (BET)1 nitrogen adsorption isotherm. The functional group present in silicates with the greatest surface acidity corntent is the silonol proop (--SiOH), which If ligniftffntly deprotonoted at physiologic values of pH end therefore will coordinate many available metal eationa (12). Random parting and fracture of fibers during the collection and processing of the silicate ahould position structural component* at the surface in proportion to quantities in tba crystal lattice. Therefore, surface ailenol density ahould parallel per centage SiOt within the orystel. Percentage SiOt of the filieatea was quantified after fusion {UBOj^LIB^OTi l"-1, w/w) at 1000*C. The melt was quenched in a mlr of HtOj, HNOj, end tartaric acid. Silicon wo# measured in duplicate specimens using inductively cooplad plasm* spectroscopy (Perkin Elmer P2000). Surface allanol concentrations were determined In triplicate specimens after wrehing 0,600 g of the fiber tarice with 60 ml of 2 V HC1 for 30 min at room temperature.Dissolution oftha dust during thia time was meeaured by atomic absorption ae release of silicon into tha supernatant and was negligible for all three Ghera. The dusts ware csntrifijgsd, washed In distilled water, and suspended in 0.01 N N&OH. Thia colloidal suapansion was agitated for 1 h end centrifuged at 1200g for 10 min. Three drops of 196 phenolpbthiloin in iaopropenal were added to the supernatant and this was titrated to a cotoriese endpoint with 0.010 U HC1 and the volume oftltrant compared with that required to neutralise the aame volume of0.01 N NaOH which had not been exposed to dust (13). Surface concentration* of Iran. Surface iron wea measured by ex posing 30.0 mg of amoslte, crocidolite, and chryaoti)e to 0.3 M sodium citrate. 1 M sodium bicarbonate, and 100 mg dlthioaite. Thia ooUoidai suspension was agitated in a water bath at 70'C for 30 min, cantxi&gad at 1200a for 10 min, end the supernatant swayed for iron in triplicate specimens by a apactrophotomctric method employing 1,10-pbaainthroline. Surface iron after intrapleural injection. To evaluate complexstion of iron by fibrous rilkatei after tntraduction into a living system, Sprague-Dawley rata (Charles River Breeding Labe, Wilmington, MA) were intrapleurelly injected with 30 mg of either smoaite, crocidolite, or chrysotile (six rats/ailicate) while under anaatheeii with halothane (2-5%), Ninety-six hours later, rata wan ansathatixad and exsangui nated, the pleural cavity was iavsged with 10 ml saline, and the hugs were excised- Collected fluid and tiaaua war* digested with 5.23% (w/v) NaOCl (50 ral/g tissue) and passed through Altars with a pore sin of 0.45 utn (MiUipore, Bedford. MA). Accumulated dust was washed 10 times with water and surface iron was assayed in triplicate employing the citrats-bicatboneta-dithionita method. Surface and total iron aftersaturation withferric chloride. To simulate the in uiuo eSact of increasing aurfeoe iron and to examine any conse quences this his on oxidant gsnsrmtion end DNA strand breakage, the surfscss of fibrous silicate* were saturated with a feme salt Amoeite, crocidolite, and chryootilo were exposed to 1 mM Fade and the suspen sions agitated for IS min. Solutions were used immediately after prep aration. Approximately 1% of Ft7* in the ferric chloride solution pre cipitated out as insoluble oxyhydroxide* within 1 h and contamination of the silicates with these compounds is negligible. Suspensions were centrifuged, washed with distilled water 10 limes, end tha fibers were examined for surface iron in triplicate. Oxidantgeneration and eccDNA atrand breakage by thoaa silicates with increased eurfaco concentration* of complexed iron were aba measured. - Total iron in crocidolite wu determined prior bo and after exposure to 1 mM FeCh for 15 min. Croddnlita 50 mg was digested in 3.6 If HjSO, and 48% HF at 100*C for 30 min (open}. This reaction was quenched with 10% boric acid and Iron measured, after reduction with dlthiamta, in triplicate using.a apectrophotomstriu method employing 1,10-phen- anthroUns. ... Oxidant generation, Iron hoe a capacity to transfer electrons and generate Oxidants (14), Such catalysis in the presence of 2-deoxy-Criboea yields maloodlaldehyde-lik* products which can be measured aa thioberinturic (TBA) reactive products (IB). The reaction mixture con tained 1 mMdsoxyriboae. 1 mM H,Ot, 1 mM ascorbate, andajther 1 mg/ ml silicate or 1 mg/ml silicate following exposure to FeCU- In an attempt to inhibit radical production, the hydroxyl radical ocevengsr dimethylthioures (DMTV) and the iron chelator deferoxamine were added with fir,si corweottationa of 20 mM end 2J3 mM, reapectivaiy, For ccmporieon with emphiholes -*r.emipg iron coupled with oxygen within the structured lattice, iron oxides were similarly tested for a capacity to generate oxidants. FeO, Fe,0. and F*jO, (1 mg/ml) ware included in reaction mixtures with deoxyriboae, KsO*, end ascorbate. The suspen sions ware incubated at 37*C for 1 h with agitation andthen centrifuged at 120Qg for 10 min. One milliliter ofboth 1.0% (w/v) TBA and 2,8% (w/v) trichloroacetic arid ware added to LO ml of supernatant, heated at JOO'C for 10 min, cooled in ice, and tha chromophore determined in triplicate specimens by it* absorbance at 632 nm. . DNA strand break*. Following exposure to oxidants, covalently closed, circular DNA (eccDNA) ia converted to OH open circular form (18). Ethidium brounds binds the latter iutercaletively with a reauhent increase In fiuoreacenca Intenaity, A suspension of 2J5 pg negatively supetcoUtd PM2 bacteriophage oocDNA (Boehringsor Mannheim, In dianapolis, IN), 1 mM H|0*, 250 eg fibrous silicate, or 250 eg fibmu silicate following exposure to FeCU. and 00. mM ascorbate was incubated for 1 h at room'temperature and centrifuged at 10,000g for 10 a. In an ottemptto inhibitDNA atrendbreakagt, tbs hydroxyl radical scavenger DMTU aod tha iron cbeletor deferoxamine were added with final con centrations of20 and 5L5 mM, respectively. FsO, Fe,0,,, end FejO, (250 pg) were similsriytested fora capacity to induce cccDNA breaks. Ethid ium bromide (2Ji pg) wea added to 250 pi of the supernatant. This woe boated at 96C for 4 min, cooled in a 26*C water bath, and the fluores cenos measured with excitation st 625 nm and emission at 00 nm. Native PM2 cccDNA was defined to exhibit a relative fluorescence value of0%. Calfthymic DNA served aa a positivecontrol (relative fluorescence of 100%). Measurements were made in replicates of five. Statmtiae. Date ere expressedAamean standard deviation.Analysis of variance was ueed to determine diSsrences between multiple group* (17). When F ratios were significant, means ware compared using Dunetn'S Multiple Range Test (18). Significance wee assumed at P < 0.06. RESULTS The amphlbole fibers amosite and crocidolite bed lower surface areas, but a higher percentage. Si04 relative to chrysotile (Table I). These values approximate reported values of SiOt for amosite, crocidolite, and chrysotile (19)The remainder of amosite and crocidolite includes iron (29.8 and 33-4%, respectively), sodium (0,0 and 4.9%, re- TABLE I Surface Area, Percentage SiO,, and Surface SUanol Density - of Fibroue Silicedea Silicate Surfoos area Surface aibuiol density (mVg) Percentage SiO* (jjreups/nm1) 1 Abbreviations used: BET, Brunaur-Emmvtt-TsU; TBA, thloborbituric; DMTO, ditncthylthioures; eccDNA. covalently closed, circu lar DNA. Amosite Crocidolite Chrysotile 2.3 0.4 8.7 1.0 28.8 1.4 49.2 0.5 48.5 0.3 39.8 0.9 7.6 IB 4,7 0.6 1.0 0.1 RECEIVE? T!ME J-JN. 20. 1 * :32AM sOT TIME JvH. 20. '1:42AM few. 06/20/02 11:33 FAX 817 735 2283 I'NT HEALTH SCIENCE DNA STRAND BREAKS AND ASBESTOS @005 15 3 X 3 ao o 5 v> Amosite Croctoollt* ChrysoHte FIG- ! Concentration* of iron contptarod to the miftt* of fibrou* ailkale*. There were (jgiuflcant differences among the three fibroin *11jeAten. Crooidolit* had the fUMUticoncentration of*urface-comple**d iron while, despite the hlchcat lutfcce ere*, chtyeocile had the hunt. Aaiostta Ciocklollta ChryeoW* FIG. 2. Surface ooBipUxoiion ofbody *oure* of iroh by aiiicafc# dojta. After introduction into an animal, fibers completed further concentra tion! of iron. Significant difference* wore found emon* ail throe dusti, with crocidolite adaothing the greatest concentration! of iron and ehryeotOa the least. sportively), and magnesium (14.5 and 0.0%, respectively), while chiyeotile has magnesium within its brucite layer (26.3%). Consistent with the measured percentage SiQj, the concentration of surface silanol groups was higher in amosite and corcidolite relative to chrysotile (Table I). Weathering in the inorganic environment of numerous primary minerals, including olivine, pyroxenes, biofcite, magnetite, ilmenite, iron sulfides, and iron carbonates, makes Pe3* and FeI+ available to the silicates for complexation (20). Using the citrate-bicarbonate-dithionita assay, significant quantities of iron were found on all three dusts (Pig. 1). The concentrations of chelatahle iron on both amphibole8 were greater than that on chrysotile. These quantities may reflect some mobilization of lattice iron in addition to metal coordinated at the surface (21). With in vivo exposures, fibers accrue iron and protein to produce ferruginous bodies. In addition, cells which phagocytese fibers demonstrate a rapid accumulation of iron (22). Both observations suggest that the surfaces of silicates are not saturated with the metal. Surface-com- Differences between the fibers in TBA-ieactive prod ucts paralleled the concentrations of surface complexed iron (Fig. 4). DMTU and deferoxamine diminished TBAreactive products so that the concentrations approached values found in-mixtures without fibers (Asa; s 0.064 0.004). The absorbances observed for oxidized products in the presence of FeO, Fe20(, and FeaO* did not differ significantly from control reaction mixture* with no fibers included. Ampbibole exposure also resulted in a higher frequency ofcccDNA strand nicks than that observed with chrysotile exposure (Fig. 5). Similar to previous investigation (4), no inctease in cccDNA strand breaks was observed on .incubation with fibers in the absence of ascorbate. This suggests that concentrations of ferrous ion complexed to the fiber surfaces were small. DMTU and deferoxamine diminished DNA strand breaks to values not significantly different from cccDNA without fiber exposure. Compa- plexed iron can be of importance in the induction of dis ease after silicate exposure only if the boat cannot elim inate the coordinated metal. After intrapleural injection, 350 the three fibrous silicates complexed in vivo source* of 'ion (Fig. 2). Again, the concentrations of surface iron were greater on the amphiboles compared to chrysotile. To delineate the effect of increased surface complexed iron observed after in utuo exposure, amosite, crocidolite, and chrysotile were saturated with Fes+. Greater concen trations of Fe3+ were necessary to saturate the surfaces of amosite and crocidolite (Fig- 3). Amphiboles have higher concentrations of Lattice iron than chrysotile and these differences in structural metal could theoretically account for variation in the associations of asbestos with oxidant generation, genotoxicity, and cancer. There was no difference in the total iron"of crocidolite prior to (3l.81.6%) and after (30.32.1%) saturation of the sur face with FeClj. Amosite CractdoHte OmrsoUs FIG. 3. Surface camplexAtlon of Inorganic iron by fibroui eiUcaree. Tbs throe fiber* adsorbed Pa** from a solution of 1 mM ferric chloride over 15 roln. Significant differences were demonstrated between the amphibole asbestos fibers and chrysotile. Difference* between the am phibole fibers were nor lucmiicant. iRECElVED T:MEBJJN. 20.11 PRINT TIME' -Uh. 20. *1 1:42AMf 06/20/02 11:35 FAX 817 735 2283 UNT HEALTH SCIENCE 16 e 5 V> 8 -Q OHIO 8T AL. Si 008 FIG. 4- Genuretion of oxidant* by fiber* mcaturod u TBA-raactive FIG. ft. Generation o oxidant* by fiber* after saturation of surfaces products ofdeoxyriboee. There were oicaificiint diffsrehcee between the with boa. The unpliibole fiber* produced (treater concentration* of ox fibrous silicctm with crocidolite and amoeite having greater absorbance ' idants,as ^fleetedby the absorbance at S32 run ofTBA-reectiv* products et 332 taa, rofloctin* higher concentrations of oxidative products of of dtoxyiibose, after saturating the surfaces with Fa1*. Differences be deoxyriboee. Differences between the amphibole fibers were not signif tween the amphibole fiber* were not significant. icant. Both dimethylthlourea (DMTU) and deferoxamine significantly diminished oxidant products of demeyribove after amoeite. crocidolite, and chiysorile exposure. with ferric chloride paralleled increments in both surface iron and oxidant generation <Fig. 7). rabie to oxidant generation, FeO, FeaOa, and FejO* did not significantly increase the frequency of cccDNA gtrand nicks above control values. While saturation of the fiber surface had no effect on the total iron, TBA-reactive products of deoxyriboee were greatly elevated again supporting a capacity of surfacecompleted metal to catalyze electron transfer (Fig. 6). Disparities between the fibers in oxidant generation prior to and after saturation of the silicate surface with ferric chloride coincided with surface Fea+ concentrations, sug gesting that those dusts which accumulate iron after they are retained within an organism may have an increased capacity to produce free radicals. Finally, increased nickmg of the cccDNA after saturation of the silicate surface DISCUSSION Major compositional differences between the asbestos fibers.include (0 structural iron oxides and magnesium oxides within the lattices ofthe amphibolea and cbiyaotile, respectively, and <il> a higher percentage of SiOz in amosite and crocidolite. Increasing the concentration of surface-complexed iron, while not altering the lattice metal content, greatly enhanced oxidant generation -and cccDNA strand nicks. Although amoeite and crocidolite have iron included in the crystal lattice, chiysotile has none in its ideal molecular formula. Isomorphic substi tution of Fei+ for Si<+ can account for the small quantity of iron in the lattice of this serpentine silicate. To mobilize this metal, there must be either dissolution of the silicate SO FIG. 5. DNA attend break* multing from che exposure af cccDNA to fibrous Ailicate*. incubation# with xmosite and crocidolite wore as sociated with significantly mote DNA strand break* thus ohiysotileDifferenees between the amphibole fibers were not significant, DMTU end deferoxamine bath sbpiUicaritly diminished cccDNA strand break* after amoeite. crocidolite, and chrysotile exposure. M) cracMeM* Ckryeeuia FIG. 7. DNA stnmd break* reiultinx from the exposure of cccDNA to fibrous silicatee after saturation of surfaces with iron. Incubations with nmosite end crocidolite were associated with significantly more DNA strand breaks than chiyaotile after saturation of their surface* with iron. Differences between the amphiboles were not significant. RECEIVED TIME JIA 20. r.:52AK 30~ TIME JJN. 20. 11:41AM 06/20/02 11:35 FAX 317 735 2283 UNT HEALTH SCIENCE 121007 DNA STRAND BREAKS AND ASBESTOS 17 or an entry of a chelator into the lattice. Using [Si} as an indicator of dissolution of the silicate, no solubilization of the dust wa* noted. However, it is unlikely that a lowmolecular-weight metal chelator could enter a crystal lat A,tice which has interatomic distances of less than 1 excise an iron cation bound within the lattice by strong crystalline forces, and exit the particle with the metaL plexed iron predicts a carcinogenic potential in humans which is promoted by observations of osteosarcomas and hematopoietic neoplasms after intravenous injection of aSFoClj (31). In farther support of a role for this metal in genotoxocity, increased iron stores in humana elevate the risk of cancer in epidemiologic studies (32). The endocytosis of the fiber is associated with oxidant Furthermore, iron oxides with the six coordination sites generation and therefore should increase with the con ofthe metal occupied, accordingly mimicking the state of centration of complexed iron and surface area (33, 34). the lattice iron in amosite and crocidolite, catalyzed nei Such endocytosis positions the silicate surface with tbe ther electron transfer nor strand breakage. This evidence complexed transition metal in the proximity of DNA and indicates that structural metal ia unlikely to participate iron may be transferred to the DNA, which also can co in free radical production in the in vitro systems employed. ordinate this metal (36, 36). Subsequent oxidant gener However, structural iron can catalyze heterogeneous ation could then result in strand breaks, genotoxicity, and electron transport. Structural oxidation of iron silicates cancer (37). A synergistic interaction between cigarette cecum through an electron bopping mechanism. This can use and asbestos fibers in genotoxicity and induction of produce an increase of ferric cation in the lattice by re bronchogenic carcinoma is anticipated as a result of in duction of surface Fea+ (23). The surface iron can then creased concentrations of lung iron available to be com be reoxidized by atmospheric 0% (29). Certain compounds pleted by tbe fiber after smoking (38-40), The results of can also complex Fa2* and accelerate mineral oxide dis these investigations predict that, for a specified fibrous solution (24-26). This can facilitate inner sphere electron silicate, those particles which are longerand thinner, rel transfer between structural and adsorbed iron states. Both ative to short,, thick fibers, will have greater values of of these mechanisms of electron transfer are ultimately both surface area and oxidant production with an in dependent on the concentration of surface-complexed creased probability of endocytosis and therefore the po iron. The higher percentage of SiOj in amphibolss and tential to induce DNA injury. Consequently tbe two pos the consequent greater density of surface silanot groups tulate* of cancer induction by fibrous silicates focusing in these fibers result in an increased capacity to coordinate on particle geometry and oxidant generation can be con metal cations at the surface relative to ehrysotile. Chry- ciliated (1,3, 4). sotile would be expected to have the highest number of Based on our results, the carcinogenic potential of a silanol groups per gram of dust of the three fibers ex fibrous silicate should be predicted by either surface con amined (Table I). The low amounts of iron on the surface centrations of silanol groups, complexed [Fe3+], or in vitro of ehrysotile even after exposure to FeCla (Figs. 1 and 3) oxidant generation after saturation of the dust with an indicate that the silanol densities (number of --SiOH per iron salt. The development of synthetic fibers with di unit area) are more important than the absolute number minished carcinogenic potential will necessitate decreas of ligand groups in determining the amount of metal co ing the number of surface functional groups with the ca ordinated. The complexing groups must be close enough pacity to complex iron and other transition metals which to each other on the mineral surface that a bi- or multi- assume two stable valence states and can transfer elec dentate complex can be formed with the iron. .. trons. After introduction into a living organism, the three fi bers completed body sources of iron onto their surfaces. ACKNOWLEDGMENTS Disparities in the concentration of surface complexd iron between the amphiboles and ehrysotile correspond with We Hunk Dr. Mike Clark of Cyprus Minerals for assistance in surface an* determinations uui Dr. 3, W- Stucki for guidance in surface iron both oxidant generation and cccDNA scission, supporting measurements, 'rfais study wo* supported in part by grants from tbe a role for iron-catalyzed oxidant generation in the geno- - National Institutes of Health (HI/-02656) end tbeNorth Caroline Luo# toxicity of fibers. DNA injury in C3Hl0Ti,/j cells after Association. exposure to crocidolite was inhibited by iron chelators, possibly reflecting a dependence on the availability of in completely coordinated iron. (27). Products of oxidation reactions catalyzed by several other iron complexes are similarly mutagenic (28). Bleomycin, a glycopeptide-employed as an anticancer drug, also coordinates iron and generates oxidants which cleave DNA (29). Such strand scission is inhibited by iron chelators (30). DNA damage after exposure of a microorganism to'hydrogen peroxide was similarly dependent on both reducing equivalents and REFERENCES 1. Stanton, M. F-, LayartJ, M-, Tegeris, A., Miller, El, Mey, M., Morgan, H,, and Smith, A. (1981) J. Natl Cantor Insfc 7.866-975. 2. HeSterbsn, T. W., and Barrett, J. C. (1985) Caranontmta 6, +73 +75. 3. Weinberg, R. O. (1968) Lancet 1,1399-13+0. . +. Lund, L. 0., and Aust, A. E. (1992) Cartfitcgerut.it 13,637-6+2. 5, Schindltr, V. W., and Stumm, W. (1997) in Aquatic Snrfaoo Chem istry. Chemical Processes at the Particle-Water Interface (Stumm, an availability of iron species (7). DNA scission by com- W., Ed), pp. 83-110. Wiley, New York. liECEIVEJ TjWJUK. 20. | 1:32AI 'PRINT OE"MUN. 20. *11:41 AN? 06/20/02 11:30 FAX 817 735 2283 VNT- HEALTH SCIENCE @ 008 18 OHIO E7T AL. 6. Hutchinson, F. (3985) Progr. Nucleic Acid Ret. Mol BfoL 22,115 2^. Addison, C, C., Addison, W. W,, Neal. G. H,, andSharp, 3. H. (1962) 164. J, Chen. Soc. 1468-1471. 7. Itaky, i. A.. and Linn. S. (1988) Science 240,1302-1309. 24. Baumtaitnar, E., Blue, M- A, Marinorich, H. and Marto, A. J. S. Crawford, D., Zbieden, )-, Amstsd, P., and Cerutti, P. (1968) On- C. (1963) Inert. Chen. 22,2224-2226. coatrne 3, 27-314. 9. Heincr, N. H., Janssen, Y- M., and Moiamim, B. T. (1991) Pmc. Noti Acad. Set USA *0. 3299-3303. 10. Mowunan, B. T,, Blgnon, J.. Corn, M., Saaion, A., and Gee, J. B. L. (1990) Science 247, 294-301. 25. Blau, M. A., Borghi. J5- B.. Marto, A. J. C,, und Regoztoni, A. E. (1964) J. Colloid Interface Scl 98,293-300. 26. Borghi, B. B,, Ragatseni, A- ., Marto, A. J. G,, and Blais, M. A .. (1989) J. Colloid Interface Set 130.301-310. ,, 27. Tutver, C. J,, and Brown, R. C. (1987) Sr. J. Cancer 66,133-138 . 11. McDonald. >i. C., and McDonald, A. D. (1987) in Aabaaux> Related 28. Tullius. T. D.. and DombrosH, B- A. (1965) Science 030,679-68). Malignancy (Annnan. KL, and Alancr. J., Eds.), pp. 87-79, Gtone 29. Sausville, E. A., Peiaach, J., and Horwiti, 5. B. (1978) Biochemistry and Stratton, New York. 17,27*0-2746. 12. Schindler, P. W. (1881) in Adsorption ofInorganic* at Solid-Liqnid 30. Bymas, R. W.-, and Petering, D. H. (1991) fliochem. Pharmacol 41, Interfaces (Anderson, M. A., and Rubin, A. J,, Ed*.), pp. 1-iB/Ann 1241-1248. Arbor Science, Ann Arbor, Ml. 31- Laisaui, J. A., Burlington, H-, Cronkiba, El P-, and Belncfce, U. 13. Arhland, S., Greathe, J., and Horan, B. (I960) Acta Chon. Stand. 14.1069-1076. 14. Graf, E.. Mahoney, J. R., Bryant, R. G., and Eaton, J, W. (1984) J. Biol Cham. 2Sd, 8620-3624, 13. HalUwell,B,, andGutteridg., J.M.C. (1981) FJEB5Lett. 128,347 352. 16. Lown, J. W. (1984) Methods Encymol 105,632-689. 17. Colton, T. (1974) Statistics in Medicine, Little, Brown, Boston. 18. Duncan, D. B. (1966) Biometrics 11,1-42. (1977) Cancer (Us. 37,3845-8560. 32. Stavana, R. G., JonM, Y,, Micozsi, M. 6., and Taylor. P. R. (1988) tf. Engl J. Med. 319,1047-1062. 33. Ohio, A- J-, Kennedy, T. P-, Whorton, A. R-, Cnwahliu, AL, Hatch, G. ., and Holdal. J. R. (1992) Am. J. Physiol 003. L51I-L516. 34. Hobeon, J,, Wright, J. L-, and Churg, A. (I960) FA$B J. 4,3135 3139. 36. Barry, B. E-, Wong, K. C., Brody, A R-, and Clapo, 3, D. (1383) *p. Lung Pei. 5,1-22. 36- Andronikashvili, E. L., Moaulishvili, L. M-, Balokobiiald, A I,, 19. Deer, W. A., Howie, R. A., and Zuxsmen, A. (1966) An Introduction Kharsbadic, N. E., Tcvgieva, T. K, and Efremova, E. Y. (1974) to the Rock Forming Mineral!, Longman Scientific Sc Technical, Cantor Bet. 34, 271-274. New York, 37. Ubbus, S. L., Illenye, S. A., and Craighead, 3. E. (1989) Cancer Re*. 20. Murad, E., end Fischar, W, R. (1988) in Iron in Soils and Clay 49,5713-5718. Manuals (Stiicki, J. W., Goodman, B. A., and Schsraitmann, U., 38. SeiikoE, L J,, Hammond, E- C,, and Churg, 3. (1908) J. Am- Med. Ed*.), pp. l-ia, Reid*), Boston- Assoc. 004.104-112. 21. Borggaerd, O, K. (1988) in Iron in Soils and Clay Minerals (Stucki, 0. W_, Goodman, B, A-. and Schwertmann. U., Eds.), pp. 83-98, Raidal, Boston. 39. Jackson, ,T. H., Schraufinaoer, L U,, Hyelop, P. A, Vaabeok, K-, Sauerhebar, R.. Weitzmon, 5. A, and Cochrane, C. G- (1987) J. Clin. Invest 80.1090-1095. 22. Kocrten, H. K., Bredaroo, P,, Glnsel, L. A., and Daevut, W. T. (1984) 40. Church, D. F., Buiiojy, T. J., and Pryor, W. A. (1990) Methods Eur. J. CM. Biol 40, 25-36, Enxymal 186(Pait B), 665-869. 1 i ( 1 RECEIVED 'IME jjN. 20. ;1:32AM PRINT TIME JUN. 20. :41AM o 6 2u. u: 11:.'!.') FAX S17 7.')5 2222S3 t-'NT HEALTH SCIENCE 002