Augmentation of Pulmonary Reactionsto Quartz Inhalation by Trace Amountsof Iron-containing ParticlesVincent Castranova,l Val Vallyathan,' Dawn M. Ramsey,2Jeffrey L. McLaurin,2 Donna Pack,1 Steven Leonard,1 MarkW. Barger,1 Jane Y.C. Ma,1 Nar S. Dalai,3 and Alex Teass21Health Effects Laboratory Division, National Institute for OccupationalSafety and Health, Morgantown, West Virginia; 2Division of Biomedicaland Behavioral Science, National Institute for Occupational Safety andHealth, Cincinnati, Ohio; 3Department of Chemistry, Florida StateUniversity, Tallahassee, Florida

Fracturing quartz produces silica-based radicals on the fracture planes and generates hydroxylradicals (OH) in aqueous media. 'OH production has been shown to be directly associated withquartz-induced cell damage and phagocyte activation in vitro. This 'OH production in vitro isinhibited by desferrioxamine mesylate, an Fe chelator, indicating involvement of a Fenton-likereaction. Our objective was to determine if Fe contamination increased the ability of inhaledquartz to cause inflammation and lung injury. Male Fischer 344 rats were exposed 5 hr/day for 10days to filtered air, 20 mg/m3 freshly milled quartz (57 ppm Fe), or 20 mg/m3 freshly milled quartzcontaminated with Fe (430 ppm Fe). High Fe contamination of quartz produced approximately57% more reactive species in water than quartz with low Fe contamination. Compared toinhalation of quartz with low Fe contamination, high Fe contamination of quartz resulted inincreases in the following responses: leukocyte recruitment (537%), lavageable red blood cells(157%), macrophage production of oxygen radicals measured by electron spin resonance or

chemiluminescence (32 or 90%, respectively), nitric oxide production by macrophages (71 %),and lipid peroxidation of lung tissue (38%). These results suggest that inhalation of freshlyfractured quartz contaminated with trace levels of Fe may be more pathogenic than inhalation ofquartz alone. Environ Health Perspect 105(Suppl 5):1319-1324 (1997)

Key words: Fenton reaction, silicosis, reactive oxygen species, inhalation, iron, nitric oxide,pulmonary inflammation, silica

IntroductionOccupational exposure to crystalline silicacan result in the development ofpulmonaryfibrosis. Silicosis may develop rapidly (1-3years) in the acute form of the disease.Acute silicosis is characterized by dyspnea,fatigue, cough, weight loss, alveolar

This paper is based on a presentation at The SixthInternational Meeting on the Toxicology of Naturaland Man-Made Fibrous and Non-Fibrous Particlesheld 15-18 September 1996 in Lake Placid, NewYork. Manuscript received at EHP 26 March 1997;accepted 23 April 1997.

Address correspondence to Dr. V. Castranova,PPRB/HELD/NIOSH, 1095 Willowdale Road (M/S2015), Morgantown, WV 26505. Telephone: (304)285-6056. Fax: (304) 285-5938. E-mail: vicl cdc.gov

Abbreviations used: AM, alveolar macrophage(s);CL, chemiluminescence; ESR, electron spin reso-nance; 'OH, hydroxyl radical(s); 02-, superoxideanion; PBN, N-tert-butyl-phenylnitrone; PHPA,4-hydroxyphenylacetic acid; RBC, red blood cell(s);'Si, silicon radical; Si-O, silicon oxyl radical.

lipoproteinosis, and diffuse fibrosis (1). Incontrast, chronic silicosis develops over aperiod of 20 to 40 years. It is characterizedby nodular lesions containing collagen in aspiral arrangement and may progress tomassive fibrosis in which restrictive lungimpairment is evident (2,3).

Cellular injury and tissue damage arebelieved to be important steps in the devel-opment of silicosis (4). This damage mayresult directly from the cytotoxicity ofquartz or indirectly from oxidant speciesproduced by silica-activated pulmonaryphagocytes (5). Such oxidants may indudesuperoxide anion (02-), hydrogen peroxide(H202), hydroxyl radical ('OH), nitricoxide (NO'), and peroxynitrite (6,7).

Acute silicosis is associated with thegeneration of freshly fractured quartzdust in occupations such as sandblasting,rock drilling, tunneling, and silica milling.

When crystalline quartz is fractured, silicon(CSi) and silicon oxyl radicals (Si-'O)respectively are formed on the fractureplanes (8). On contact with aqueousmedia, these surface radicals generate 'OH(9). The Fe chelator desferrioxamine sig-nificantly reduces this 'OH production byfreshly fractured quartz in water (10). Thissuggests that trace contamination of quartzwith ferrous Fe can enhance 'OH genera-tion via a Fenton-like reaction as follows:

Si-'O+ H20 ->SiOH + 'OHSi-'O+ 'OH -*SiOOH

SiOOH + H20 -SiOH + H202Fe2+ + H202 -*Fe3+ +'OH+OH-

In vitro, freshly fractured quartz causesgreater peroxidation of membrane lipids,greater membrane leakage, and a greaterdecrease in cell viability than aged quartz.Freshly fractured quartz is also a morepotent stimulant of reactive species pro-duction by alveolar macrophages (AM) invitro (9,11). In comparison to aged quartz,inhalation of freshly milled quartz resultsin a significantly enhanced cytotoxicity(lipid peroxidation of lung tissue andlavage levels of red blood cells [RBC], pro-tein, and lysosomal enzymes) and elevatedinflammation (lavageable neutrophils, gen-eration of oxidant species from AM, andhistological scoring of lung infiltrates)(12,13). Evidence suggests that a directrelationship exists between 'OH generationand cytotoxicity, as the production of both'OH- and quartz-induced biological reac-tions decrease in a similar fashion withtime after fracturing (9,11).

Because Fe enhances 'OH generationby freshly fractured quartz and a relation-ship exists between 'OH production andthe cytotoxic potency of quartz, the ques-tion was whether trace amounts of Fewould increase the pulmonary responses toinhaled quartz. To resolve this question,we conducted an inhalation exposure inwhich rats were exposed to filtered air,freshly milled quartz with low Fe contami-nation, and freshly milled quartz contain-ing 7.5 times more Fe contamination. Lowand high Fe contamination of quartz wasachieved by altering the length of the poly-ethylene delivery tube of the stainless steelscrew feeder. The following pulmonaryresponses to quartz inhalation (20 mg/m3,5 hr/day, 10 days) were monitored 1 day

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CASTRANOVA ET AL.

postexposure: lavageable RBC, AM, andleukocytes; production of reactive speciesfrom AM; and lipid peroxidation oflung tissue.

MethodsExperimental DesignMale Fischer 344 rats (7 weeks of age) weredivided into three groups of 20 rats each(filtered air controls, freshly milled quartzwith low Fe contamination, and freshlymilled quartz with high Fe contamination)and housed in two 10 m3 Hinners-typewalk-in chambers (quartz exposed) or a5 m3 Hinners-type chamber (air control).Freshly milled quartz was generated in anair jet mill (Trost Gem-T Research ModelType 1047 [Garlock Inc., Newtown, Pa]fitted with a polyurethane liner; P-jet pres-sure of 70 psi and an 0-jet pressure of 100psi). Low and high Fe contamination ofquartz, i.e., quartz with low Fe contamina-tion and quartz with high Fe contamina-tion were achieved by adjusting the lengthof the screw feeder delivery tube (6.35 vs15 cm to produce low vs high Fe contami-nation, respectively). Rats were exposed5 hr/day for 10 days and were sacrificed1 day postexposure for determination ofbiological responses. Details of the quartzdose, particle diameter, and trace metalcontamination for the exposure groups aregiven in Table 1. Note that both quartzinhalation groups were exposed to particlesof the same size (2.0-2.1 pm) and at thesame dose (21-19 mg/m3). The major dif-ference between the two quartz aerosolswas their trace metal content (57 ppm Fefor low Fe contamination and 430 ppm forhigh Fe contamination).

Generation ofFreshly Milled SilicaFreshly fractured a-quartz was preparedfrom a stock IOTA lot 11383 quartz sam-ple obtained from Unimin Corporation

(Spruce Pine, NC). This stock quartz hada mass mean diameter of 191 pim and wassemiconductor grade in purity, i.e., greaterthan 99% pure crystalline silica (SiO2) asdetermined by X-ray diffraction analysisunder scanning electron microscopy andoptical microscopic examination underpolarized light. Freshly fractured a-quartzwas prepared as follows: stock of quartzdust was supplied by an AccuRate Series100 dry material feeder (AccuRate,Whitewater, WI) with a 6.4-mm diameterstainless steel helix and a polyethylenedelivery tube to a Trost Gem-T ResearchModel Type 1047 jet mill (Garlock,Newtown, PA) fitted with a polyurethaneliner and stainless steel jets and operatedwith dry, charcoal-scrubbed compressedair at a P-jet pressure of 480 kPa (70 psi)and an 0-jet pressure of 690 kPa (100psi). The difference in trace metal contentof the two quartz aerosols is due to stain-less steel contamination from the drymaterial feeder. The longer the stockquartz remained in contact with the stain-less steel helix, the higher the resultingcontamination. Shortening the length ofthe polyethylene delivery tube shortenedthe contact time, thus lowering the stain-less steel content of dust going to thejet mill.

Inhalation ExposureThe air supply to the animal chambers wasrough filtered; chilled or heated to 16°C;reheated to 23°C (feedback control fromthe chambers); passed through a high effi-ciency particle filter, a charcoal bed, and amedium efficiency filter; and rehumidifiedwith a steam humidifier. Air flow throughthe animal chambers was 12 air changes/hrand pressure in the chambers was held to0.25 cm of water less than in the labora-tory. The aerosol of freshly milled a-quartzwas passed directly from the air jet mill via16 and 19 mm (inside diameter) tubing

Table 1. Characteristics of exposure aerosols.

Group SI dose,a mg/m3 Particle diameter,b Pm Iron,c ppmAir control 0Si, low Fe contamination 21 ± 0.1 2.0 (1.9) 57 ±33Si, high Fe contamination 19 ± 0.1 2.1 (2.0) 430 ± 73

"Silica concentration within the exposure chambers was determined gravimetrically from filter samples. Mean ±SD (n= 54). bMass median aerodynamic diameter was determined using a nine-stage Andersen cascade impactorACFM particle-sizing sampler (Andersen Samplers, Atlanta, GA). Median value (geometric SD) (n=3). cTrace Fecontamination was determined by proton-induced X-ray emission spectroscopy performed by Element AnalysisCorporation (Lexington, KY). Mean ± SD (n= 10). Other trace contaminants were Si with low Fe contamination:16ppm chromium, 0.6 ppm copper, 1.2 ppm manganese, 7.2 ppm nickel, 1.3 ppm zinc, and 11 mg/g carbon. Carbonwas measured by Galbraith Laboratories (Knoxville, TN) (n=5). Si with high Fe contamination:107 ppm chromium,2.0 ppm copper, 11 ppm manganese, 47 ppm nickel, 0.7 ppm zinc, and 7.9 mg/g carbon.

through a BGI Aero-2 cyclone (BGI Inc.,Waltham, MA) to the mixing chamberin the air duct 46 cm upstream of theexposure chamber. To aid mixing of thetwo streams, the juncture was 3 cm down-stream of an orifice plate and 20 cmupstream of a circular baffle centered in theduct. The static charge on the dust wasneutralized by a Static Control ServicesModel PFC-20 pulse flow controller(Static Control Service, Palm Springs, CA)with a Model AN-6 ionizing air nozzlelocated about 5 cm upstream of the mixingchamber. Exposures were conducted dur-ing the animals' dark cycles (i.e., duringtheir active periods). Preliminary experi-ments with a fog generator revealed thatbaffling resulted in single-pass flow ofair through the animal cages within theexposure chambers.

Temperature (1 8.5-24.2°C), humidity(37-68%), and ammonia levels in thechambers were monitored continuously.Filter samples of the chamber atmosphereswere obtained continuously to determinedust concentration by gravimetric analysis.Additional filter samples were obtained tomonitor trace contaminants, dust activity(i.e., H202production), surface radicalsby electron spin resonance (ESR) spec-troscopy, and particle size by scanningelectron microscopy. The mass medianaerodynamic diameter of the quartz parti-cles was determined using a nine-stageAndersen cascade impactor ACFM parti-cle-sizing sampler (Andersen Samplers,Atlanta, GA).

Measurement ofSurfacRmadicalsSurface reactivity of freshly milled andaged quartz was determined by monitoringsurface free radicals using ESR spec-troscopy. ESR spectra were obtained witha Bruker Model ER300 spectrometer(Braker Instrument Corp., Billerica, MD)interfaced with an ASPECT 3000 micro-computer (Braker Instrument Corp.). Thefilter samples of dust were rolled, placedinto a quartz ESR sample tube, andinserted into the center of the ESR cavity.All measurements were conducted atroom temperature. The following settingswere used: microwave power= 13 dB,microwave frequency= 9.53 GHz, modu-lation frequency= 100 kHz, modulationamplitude = 1 G, and receiver phase= 0 or180. Silicon-based radical sites were char-acterized by ESR spectra centered aroundg= 2.0015. Surface activity was deter-mined from peak height and expressed assignal height per milligram of quartz.

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TRACE IRON-ENHANCED RESPONSE TO QUARTZ INHALATION

Measurement ofSilicaReactive ProductsGeneration of reactive products by freshlyfractured quartz in aqueous media wasdetermined by monitoring H202 produc-tion fluorometrically. The peroxidase-cat-alyzed oxidation of 4-hydroxyphenylaceticacid (PHPA) by H202 yields a strongly flu-orescent dimer ofPHPA (14). In our appli-cation, the quartz-laden filter was placedinto 10 ml of buffer solution (5 mMKNaHPO4; pH 7) and sonicated for 1 min.The filter was then rinsed with deionizedwater to remove residual dust. The suspen-sion was agitated at 25°C for 30 min toallow efficient transfer of H202 into thesolution. The supernatant was made 0.024mM in PHPA and 8.9 activity U/ml in per-oxidase and stored in the dark for 30 minto allow for reaction. The pH was thenadjusted to greater than 10 and analysiswas carried out with a flow injection sys-tem and a fluorescence detector (excitation320 nm, emission 420 nm). The systemwas calibrated with standard solutions ofH202 and measurements were correctedfor sample blank and volume dilution.

Detemination ofLavagable CellsAnimals were anesthetized and lungs werelavaged 10 times with 8-ml aliquots ofCa2+, Mg2+-free phosphate-bufferedmedium (145 mM NaCl, 5 mM KCl, 1.9mM NaH2PO4, 9.35 mM Na2HPO4, and5.5 mM glucose; pH 7.4). Lavage fluid wascentrifuged, and cells were collected,washed, and resuspended in HEPES-buffered medium (145 mM NaCl, 5 mMKCl, 10 mM HEPES, 5.5 mM glucose,and 1 mM CaCI2; pH 7.4). Cell countswere obtained using an electronic cellcounter. Cell differentials were determinedusing a cell sizing attachment and charac-teristic cell volumes to count RBC, leuko-cytes (lymphocytes and granulocytes), andAM (15).

Measurement ofAlveolarMacpha ActivityChemiluminescence (CL) generated frompulmonary phagocytes was measured at37°C in the presence of 8 pg% luminolusing a Berthold LB953 luminometer(Wallac, Gaithersburg, MD). AM (7.5 x105 cells) were suspended in 0.75 ml ofHEPES-buffered medium and preincu-bated at 37°C for 10 min prior to beingplaced into the luminometer. Zymosan-stimulated CL (counts per minute withzymosan minus counts per minute at rest)was measured in the presence of 2 mg/ml

unopsonized zymosan added just beforemeasuring CL. Note that granulocytes donot respond to unopsonized zymosan (15).NO synthase-dependent CL (counts perminute with zymosan minus counts perminute with zymosan plus L-NAME[Nw-nitro-L-arginine methyl ester]) wasdetermined as the amount of zymosan-stimulated CL that was inhibitableby 1 mM L-NAME added during thepreincubation period.

Oxygen radical generation from alveolarphagocytes was measured with an electronspin resonance spectrometer using a radicalspin trap. AM (5 x 105 cells/ml) obtainedfrom the three groups of animals wereincubated with 0.1 M N-tert-butyl-phenyl-nitrone (PBN) for 1 hr at 37°C in an oscil-lating water bath. Control experimentswere carried out without cells and withoutPBN. The reaction was terminated by theaddition of 5 ml chloroform:methanol(2:1) and total lipids were extracted. Thechloroform was evaporated in a boilingwater bath and the lipid was reconstitutedin 0.5 ml chloroform and transferred tonuclear magnetic resonance quartz tubes.On the basis of a method of Vallyathanand colleagues (16), ESR measurementsof the chloroform extracts were madewithin 1 hr using a Varian E-109 ESRspectrophotometer (Varian Associates,Palo Alto, CA) operating at X-band (9.7GHz) at the following spectrometer set-tings: receiver gain, 3.2 x 105; microwavepower, 50 mW; modulation amplitude,2 G; scan time, 100 sec; time constant,1 sec; magnetic field, 3417± 50 G. ESRspectra were recorded and integrated forthree scans as a measure of reactive oxygenspecies generated from phagocytes. Scalingand analysis of the spectra were madeusing the EPR (U.S. EPR, Clarksville,MD) DAP 2.0 program.

Measurement ofthe LungLipid PeroxidationLipid peroxidation of lung tissue from thethree groups of animals was monitored bymeasuring thiobarbituric acid reactive sub-stances generated during incubation oflung slices for 1 hr in a buffered mediumwithout any other stimulation, accordingto the method of Hunter and co-workers(17). Thin lung tissue slices (300-450 mg)were incubated in phosphate-bufferedmedium at pH 7.4 for 1 hr at 37°C in ashaking water bath. The reaction was ter-minated by the addition of 0.3 ml 5 NHCI and 0.625 ml 40% trichloroaceticacid. After mixing, 0.625 ml thiobarbituric

acid was added to the reaction mixture,mixed, and heated in a water bath at 95°Cfor 20 min. The thiobarbituric acid-reactive substances developed a color thatwas measured after cooling at 540 nm andcompared with malondialdehyde reactionwith thiobarbituric acid. Malondialdehydeproduction was calculated from a standardgraph that was made using the samereagents and known concentrations of mal-ondialdehyde. Control experiments werecarried out without lung tissues, with inac-tivated lung tissue, and in the presence ofan antioxidant (butyl hydroxy toluene) toinhibit lipid peroxidation.Statstical Ana isData are means ± standard errors of 4 to 8values determined from separate rats. Datawere analyzed by analysis of variance todetermine significance at p < 0.05.

ResultsFreshly fracturing quartz in a jet mill breaksSi-O bonds and generates 'Si and Si-O'radicals on the fracture planes. As shown inFigure 1, trace contamination with stainlesssteel particles has no effect on the genera-tion of these surface radicals. Indeed, thereis no difference in the intensity of the ESRsignal produced by quartz with low Fe con-tamination compared with that producedby quartz with high Fe contamination afterjet milling (intensity ratio of 1 and 0.93 forSi [low Fe contamination] and Si [high Fecontamination], respectively).

In contrast, once placed in aqueousmedia, quartz with high Fe contaminationproduces more reactive species than quartz

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CASTRANOVA ET AL.

with low Fe contamination (Figure 2).These data suggest that trace contaminationof quartz with Fe particles (430 ppm) issufficient to allow Fenton-like reactions tooccur and result in enhanced generation ofreactive species by freshly fractured quartz.

Bronchoalveolar lavage indicates thatinhalation of quartz with high Fe contami-nation causes significantly more damage tothe alveolar air-blood barrier than anequal exposure to quartz with low Fe con-tamination, as measured by the lavageyield of RBC (Figure 3). Freshly milledsilica contaminated with high Fe is alsomore inflammatory than milled silica withlow Fe contamination, as judged by the

infiltration of leukocytes (lymphocytes andgranulocytes) into the airspaces of the lung(Figure 3).

The activity of AM harvested fromquartz-exposed rats was determined bymonitoring: zymosan-induced chemilumi-nescence, NO-dependent chemilumines-cence, and the production of oxygenradicals using ESR and a spintrap.Inhalation of quartz contaminated withstainless steel Fe particles, i.e., quartz withhigh Fe contamination, significantlyincreases zymosan-stimulated chemilumi-nescence from AM above that seen afterexposure to quartz with low Fe contamina-tion (Figure 4). Similar Fe enhancement is

observed when NO-dependent (L-NAME-inhibitable) macrophage chemilumines-cence is monitored in response to zymosan(Figure 5) and when measuring the gen-eration of oxygen radicals by alveolarphagocytes (Figure 6).

The enhanced production of reactivespecies in response to inhalation of quartzwith high Fe contamination compared toquartz with low Fe contamination results ina greater level of lipid peroxidation in lungtissue exposed to high Fe-contaminatedquartz (Figure 7). This increased lung dam-age may explain the breakdown of the alve-olar air-blood barrier suggested in Figure 3,i.e, the increase in lavageable RBC.

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Figure 2. Production of reactive species by freshlyfractured quartz in aqueous medium. Quartz with lowFe contamination and quartz with high Fe contamina-tion contain 57 and 430 ppm Fe particles, respectively.Data represent means ± SEM from eight chamber sam-ples. *Significant increase above quartz with low Fecontamination (p<0.05).

Figure 4. Zymosan-stimulated chemiluminescencefrom rat alveolar macrophages harvested by bron-choalveolar lavage 1 day after quartz exposure (20mg/m3, 5 hr/day for 10 days). Quartz with low Fe cont-amination and quartz with high Fe contamination con-tain 57 and 430 ppm Fe particles, respectively. Dataare means ±SEM from four rats. *Significant increaseabove quartz with low Fe contamination (p< 0.05).

Figure 6. Generation of oxygen radicals produced byrat alveolar phagocytes harvested by bronchoalveolarlavage 1 day after quartz exposure (20 mg/m3, 5 hr/dayfor 10 days). Oxygen radicals are measured by ESRusing a spin trap. Quartz with low Fe contaminationand quartz with high Fe contamination contain 57 and430 ppm Fe particles, respectively. Data are means ±SEM from four rats.

AM_ RBC_ Leukocytes

n--I |Filtered air (luhrtz, low Fe Quartz, high Fe

Figure 3. AM, RBC, and leukocytes (lymphocytes andgranulocytes) harvested by bronchoalveolar lavage 1day after quartz exposure (20 mg/m3 , 5 hr/day for 10days). Quartz with low Fe contamination and quartzwith high Fe contamination contain 57 and 430 ppm Feparticles, respectively. Data are means ± SEM fromeight rats. *Significant increase above quartz with lowFe contamination (p<0.05).

Figure 5. NO-dependent chemiluminescence from ratAM harvested by bronchoalveolar lavage 1 day afterquartz exposure (20 mg/m3, 5 hr/day for 10 days).Quartz with low Fe contamination and quartz with highFe contamination contain 57 and 430 ppm Fe particles,respectively. Data are means ± SEM from four rats.*Significant increase above quartz with low Fe contam-ination (p<0.05).

Figure 7. Lipid peroxidation of lung tissue measuredas malondialehyde production 1 day after quartz expo-sure (20 mg/m3, 5 hr/day for 10 days). Quartz with lowFe contamination and quartz with high Fe contamina-tion contain 57 and 430 ppm Fe particles, respectively.Data are means ± SEM from four rat lungs. *Significantincrease above quartz with low Fe contamination)(p<0.05).

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997

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TRACE IRON-ENHANCED RESPONSE TO QUARTZ INHALATION

DiscussionThe results of this investigation indicatethat pulmonary responses 1 day afterinhalation exposure to a-quartz are rathersmall when the quartz used is of semicon-ductor purity and efforts are employed tominimize trace metal contamination by thedust generation system. The simple increasein the length of the delivery tube of thescrew feeder is sufficient to increase the Fecontamination of silica from 57 to 430ppm. Such trace Fe contamination increasesthe generation of reactive species by silica inaqueous media by 52%. Trace Fe contami-nation also results in augmentation of pul-monary responses to quartz inhalation(Table 2). That is, in comparison to quartzwith low Fe contamination, quartz withhigh Fe contamination increases leukocyterecruitment by 537%, lavagable RBC by157%, phagocyte production of oxygen rad-icals by 32%, AM zymosan-stimulatedchemiluminescence by 90%, NO-depen-dent chemiluminescence from AM by 71%,and lung lipid peroxidation by 38%.

Ghio et al. (18) have proposed thatsilica-induced lung disease is mediated bysurface coordination with Fe and the sub-sequent generation of 'OH via the Fentonreaction. Indeed, silica-Fe complexationhas been demonstrated in rat lungs afterintratracheal instillation of silica (19). Invitro treatment of silica with desferrioxam-ine mesylate (an Fe chelator) decreases sil-ica-induced oxidation of lung surfactantand the silica-stimulated respiratory burstofAM (20,21). Similarly, desferrioxaminedecreases the ability of freshly fracturedquartz to generate 'OH in aqueous media(10). Such pulmonary production of 'OHhas been demonstrated in quartz-exposedrats and has been associated with lunginflammation (22). The results of the cur-rent investigation support the hypothesis

Table 2. Increased activity of quartz with high Fe con-tamination.

Percent increasequartz, high Fe:

Parameter quartz, low Fe

Quartz reactive species 52Lavage RBC 157Lavage leukocytes 537AM zymo-stim CL 90AM NO'-dependent CL 71AM oxygen radicals 32Lung lipid peroxidation 38

Zymo-stim, zymosan-stimulated.

that trace Fe contamination can augmentthe generation of oxidants by quartz andenhance its inflammatory and cytotoxicpotency in the lung. In addition, ourresults extend the hypothesis by demon-strating that this augmentation is possiblewith particulate Fe contamination as mightbe experienced in occupational settingssuch as rock drilling or sandblasting.

In contrast to the above hypothesisthat Fe contamination increases the cytox-icity of quartz, Nolan et al. (23) havereported that Fe chloride solutionsdecrease the in vitro hemolytic potential ofquartz by neutralizing the negative surfacecharge of quartz. Similarly, Fe chloridesolutions can decrease quartz-inducedhydrogen peroxide secretion from AM invitro (24). Particulate Fe is also protectiveagainst quartz-induced pulmonary inflam-mation (neutrophil recruitment) afterintratracheal instillation of carbonyl Feplus quartz (24). This apparent conflict isresolved by an analysis of the Fe doserequired to neutralize the negative surfacecharge of quartz and result in significantdetoxification. In solution, as much as 1M Fe chloride is required to eliminate thenegative surface charge of quartz, whereasas a particulate, Fe must be 12-fold in

excess of quartz for depression of toxicityto become apparent (24). In contrast,quartz-Fe complexes in the lung are asso-ciated with only trace levels of Fe (19).Furthermore, the current investigationclearly demonstrates that trace contamina-tion of quartz with Fe particles (430 ppm)enhances pulmonary inflammation andlung damage.

In this study, the reactivity of the freshlyfractured quartz was associated with thelevel of Fe contamination. However, itmust be noted that the two quartz aerosolsinvestigated in this study really differed intheir contamination by stainless steelpartides. Thus, as demonstrated in Table 1,amounts of numerous trace metals(chromium, copper, manganese and nickel)as well as Fe were elevated in the more toxicquartz sample. Emphasis has been placedon Fe because of the extensive literatureassociating Fe with the generation of *OHvia the Fenton reaction. However, evidenceexists that chromium (Cr3+) can also gener-ate 'OH in the presence of H202(25). Inaddition, nickel (Ni2+) has been reported togenerate 'OH from hydroperoxides butonly in the presence of oligopeptides suchas glutathione (26).

In conclusion, the negative surfacecharge of crystalline quartz contributes toits unique toxicity. Gross quantities of Fecan coat the quartz surface and neutralizethis surface charge to partially detoxifyquartz. However, the required Fe:quartzparticulate ratio of greater than 12:1 is notlikely in occupational settings where silico-sis is a concern. In contrast, trace contami-nation of quartz with Fe may occur in rockdrilling or sand blasting. Such trace Fecould catalyze the generation of 'OHby freshly fractured quartz leading toincreased lung damage and inflammation,as shown in this investigation.

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1324 Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997