Environmental Research Section A 82, 245}252 (2000)doi:10.1006/enrs.1999.4025, available online at http://www.idealibrary.com on

Involvement of Oxidative Stress in Crystalline Silica-Induced Cytotoxicityand Genotoxicity in Rat Alveolar Macrophages

Zhuo Zhang,* Han-Ming Shen,- Qi-Feng Zhang,* and Choon-Nam Ong-

*Pneumociniosis Division, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; and-Department of Community, Occupational and Family Medicine, Faculty of Medicine (MD3), National University of Singapore,

16 Medical Drive, Singapore 117597, Republic of Singapore

Received April 27, 1999

Alveolar macrophages (AMs) occupy a key posi-tion in silica-induced pulmonary Abrosis, althoughthe mechanisms are yet to be elucidated. In thepresent study we examined the involvement of oxi-dative stress and reactive oxygen species formationin silica-induced cytotoxicity and genotoxicity incultured rat AMs. A lucigenin-dependent chemi-luminescence test was used to determine super-oxide anion (O2

2 ), and a 2@,7@-dichloroBuorescindiacetate Buorescence test was employed tomeasure the hydrogen peroxide (H2O2) level. Thecytotoxic and genotoxic effects caused by silica inAMs were examined by lactate dehydrogenase(LDH) leakage and single-cell gel electrophoresis(comet assay), respectively. The results showed thatsilica enhanced O2

2 and H2O2 formation in AMs.There were clear dose- and time-dependent rela-tionships in silica-induced cytotoxicity and geno-toxicity. Furthermore, superoxide dismutase andcatalase were able to reduce silica-induced LDHleakage and DNA damage, with concurrent signiA-cant inhibition on silica-induced oxidative stress inAMs. These Andings provide convincing evidencethat oxidative stress mediates the silica-inducedcytotoxicity and genotoxicity. The understandingof such a mechanism may provide a scientiAc basisfor the possible application of antioxidants inpreventing the hazardous effects of silica. ( 2000

Academic Press

Key Words: crystalline silica; alveolar macro-phage; oxidative stress; reactive oxygen species;cytotoxicity; genotoxicity.

INTRODUCTION

Silicosis is among the most common pneumoco-nioses caused by occupational exposure to crystal-line silica. Excessive exposure to these dusts result

24

in an extensive pulmonary 7brosis for which there isno effective clinical treatment. The mechanisms in-volved in the development of pulmonary silicosishave not been well de7ned. Reactive oxygen species(ROS) have been recognized as important intermedi-ates in biologic reactions and as a primary cause ofcell injury and cell death in various pathophysiologicprocesses (Halliwell and Cross, 1994). In recentyears ROS have been implicated in pulmonary dis-eases caused by crystalline silica and many otheroccupational and environmental pollutants (Val-lyathan and Shi, 1997). Such a hypothesis is gener-ally based on evidence from (i) cell-free systems and(ii) in vivo animal experiments. For instance, theformation of free radicals on the surface of silica andsubsequent generation of ROS, including hydrogenperoxide (H2O2), superoxide anion (O~2 ), singlet oxy-gen (1O2), and hydroxyl radical (zOH), all have beendetected using ESR with spin-trapping techniquesin cell-free environments (Shi et al., 1988; Val-lyathan et al., 1988). In vivo experiments have alsodemonstrated that silicotic lungs are in a state ofoxidative stress and that increased generation ofROS is associated with enhanced levels of oxidativeenzymes, such as superoxide dismutase and gluta-thione peroxidase, and lipid peroxidation(Vallyathan et al., 1995, 1997).

It is well established that alveolar macrophages(AMs) play a critical role in the 7brosis process insilicosis (Davis, 1986; Mossman et al., 1998). AMsoccupy a key position in mediating the interactionbetween inhaled particulates and various cell types,such as lymphocytes and 7broblasts, through therelease of a wide variety of in8ammatory andgrowth-mediating factors, as well as cytokines(Holian et al., 1994).

On the other hand, silica has recently been classi-7ed as a con7rmed human carcinogen (IARC, 1997).

5

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246 ZHANG ET AL.

Silica-induced DNA strand breaks have been shownin cell-free systems using j HindIII DNA, and thedamage was believed to be due to hydroxyl radical(Daniel et al., 1993, 1995). Moreover, an elevatedlevel of 8-hydroxy-2@-deoxyguanosine (8-OHdG) insilica-treated rat lung tissue has also been noted,further suggesting the involvement of silica-inducedoxidative DNA damage (Yamano et al., 1995). Never-theless, few of these studies provide evidence to re-8ect the involvement of ROS generation on thegenotoxic effect of silica in AMs.

In this study, we investigated the involvement ofROS in the cytotoxic effect and DNA damage causedby silica in AMs in vitro. The main objective was totest the hypothesis that oxidative stress mediatessilica-induced cytotoxicity and genotoxicity in AMs.

MATERIALS AND METHODS

Chemicals

Standard crystalline silica was obtained from theInstitute of Occupational Medicine, Chinese Acad-emy of Preventive Medicine (Beijing, China), witha purity of 99%. More than 95% of the particles wereless than 5 lm in diameter. The surface area for eachparticle was estimated to be around 78.5 lm2.Lucigenin and 2@,7@-dichloro8uorescin diacetate(DCFH-DA) were purchased from Molecular Probes(Eugene, OR). Superoxide dismutase (SOD),catalase (CAT), penicillin, streptomycin, andethidium bromide were all from Sigma (St. Louis,MO); RPMI 1640 medium, fetal bovine serum (FBS),low-melting and normal-melting-point agarose werefrom Gibco BRL (Life Technologies, Gaithersburg,MD).

Cell Culture and Treatment

Male Sprague}Dawley rats (body weight 200 to250 g) were provided by the Animal Center, NationalUniversity of Singapore. Rats were anesthetized byip injection of a mixture of fentanyl citrate,8uanisone (Jassen, Belgium), and midazolam (F.Hoffmann}La Roche, Switzerland). The AMs werecollected by lavaging isolated lungs with PBS afterthe animals were killed by bloodletting from thefemoral artery. Collected AMs were washed withPBS and cultured in RPMI 1640 medium, containing10% FBS, penicillin (100 units/ml), and streptomy-cin (100 lg/ml) in a 5% CO2 incubator at 373C.

The suspension of silica was prepared in PBS andonly one stock solution was used throughout thestudy. The 7nal concentration was designated inmicrograms per 106 cells. The AMs were treated

with different concentrations for designated periodsof time. The effects of SOD and CAT were examinedby pretreating cells for 3 h in FBS-free medium andwashed once with PBS.

Lucigenin}Dependent Chemiluminescence Test

Lucigenin}dependent chemiluminescence (CL) inAMs was measured as described earlier (Shen et al.,1999). The stock solution of 10 mM lucigenin wasprepared in PBS and stored at !203C in dark. Thebasic reaction mixture contained 1]106 cells and100 lM lucigenin in 1 ml PBS. The CL reaction wasinitiated by the addition of lucigenin and silica, andthe CL level was monitored as relative light units ina luminometer (Tampa, FL) every 30 s for a totalperiod of 10 min.

Analysis of 2@,7@-Dichloro>uorescin DiacetateFluorescence in AMs

The level of H2O2 in AMs was determined using2@,7@-dichloro8uorescein diacetate (DCFH-DA) asa 8uorescence probe, based on the method as des-cribed by Shen et al. (1996). The principle of thisassay is that DCFH-DA diffuses through the cellmembrane and is enzymatically hydrolyzed by in-tracellular esterases to non8uorescent dichloro-8uorescin. In the presence of ROS (mainly H2O2),this compound is rapidly oxidized to highly 8uor-escent dichloro8uorescein (DCF) (Bass et al., 1983;Lebel et al., 1992). The DCFH-DA was dissolved inabsolute ethanol at a concentration of 5 mM as stockand kept at !703C in the dark. The AMs wereincubated in 24-well plates, each well containing1.5]105 AMs and 2 lM DCFH-DA in 2 ml medium.The reaction was initiated by the addition of DCFH-DA and particles into the medium and incubated at373C up to 4 h. The 8uorescence intensity was mea-sured using a plate-reader (Tecan Spectra8uo Plus,Sweden) with excitation wavelength 485 nm andemission wavelength 535 nm.

Determination of Lactate Dehydrogenase Leakage

Activity of lactate dehydrogenase (LDH) in themedium was measured using an Abbott VP Bio-chemical Analyzer with the test kit (Chicago, IL), asestablished in our laboratory (Shen et al., 1995). Thetotal LDH activity was also determined after cellswere disrupted using ultrasonication. Lactate dehy-drogenase leakage (%) was calculated as (LDH ac-tivity in medium/total LDH activity]100).

FIG. 1. The effect of silica on O~2 formation in AMs as deter-mined using the lucigenin-dependent CL test. In the dose}res-ponse study (A), the data were expressed as an integrated value ofCL within the whole period of measurement (10 min). In the time-course study (B), cells were treated with 100 lg/106 cells silica.The basic reaction mixture contained 1]106 cells and 100 lMlucigenin in 1 ml PBS and reaction was initiated with addition ofparticles and lucigenin. Data are presented as means$SD(n"4); *P(0.05 compared to the control group (one-wayANOVA with Scheffe’s test).

SILICA-INDUCED OXIDATIVE STRESS 247

Analysis of DNA Damage

Damage to DNA was detected using single-cell gelelectrophoresis (SCGE or comet assay) according tothe method described earlier (Yang et al., 1999).Brie8y, fully frosted slides were covered with 0.7%of normal-melting agarose as the 7rst layer, a mix-ture of cell suspension and 0.7% of low meltingagarose (LMA) as the second layer, and the thirdlayer was 0.7% LMA. After solidi7cation at 43C,slides were immersed in the lysing buffer (2.5 mMNaCl, 100 mM Na2EDTA, and 10 mM Tris, pH 10,with freshly added 1% Triton X-100 and 10%DMSO) at 43C for 1 h. Slides were then placed ina horizontal electrophoresis tank 7lled with freshlyprepared electrophoresis solution (300 mM NaOH;1 mM Na2EDTA, pH 13) for 20 min. Electrophoresiswas conducted at 43C for 20 min (25 V and 0.3 A).The slides were then neutralized in neutralizationbuffer (0.4 M Tris}HCl, pH 7.5), stained withethidium bromide, and examined using a 8uores-cence microscope (Nikon, Japan). Images of 100 ran-domly selected cells from each slide were analyzed.The degree of DNA damage was graded into 7vecategories according to the amounts of DNA in thetail: grade 0, no damage, (5%; grade 1, low-leveldamage, 5}20%; grade 2, medium-level damage,20}40%; grade 3, high-level damage, 40}95% dam-age; and grade 4, total damage, '95%.

Statistical Analysis

All data were based on at least three independentexperiments and presented as means$SD and ana-lyzed using one-way ANOVA with Scheffe’s test. Theresults of comet assay were presented as percentageof grade and analyzed using the chi-squared test.A P value less than 0.05 was considered statisticallysigni7cant.

RESULTS

Effect of Silica on O~2 Formation

In the present study, lucigenin-dependent CL wasdetermined as the parameter for O~2 formation. Theresults clearly showed that silica-induced O~2 forma-tion was both time and dose dependent (Figs. 1A and1B). There are signi7cant differences between silica-treated AMs and the control from the 7rst minuteonward. The relative light unit in silica-treated AMs(100 lg/106 cells) in 10 min was nearly 7ve timeshigher than in the controls.

Effect of Silica on H2O2 Formation

Figure 2A shows the dose}response relationship ofsilica-induced DCF 8uorescence formation. Com-pared to the untreated cells, all three doses of silicasigni7cantly enhanced the DCF 8uorescence inten-sity. A time-dependent change of DCF 8uorescencewas also observed when AMs were treated with100 lg silica/106 cells. Signi7cant differences wereobserved from 0.5 h onward. At the end of theincubation (4 h), the DCF 8uorescence intensity insilica-treated AMs was two times higher than in thecontrol (Fig. 2B).

Cytotoxicity of Silica

Silica-induced cytotoxicity in AMs was evalu-ated by LDH leakage. Figure 3A shows a clear

FIG. 2. The effect of silica on H2O2 formation in AMs exam-ined using DCFH-DA 8uorescence test. The reaction took place in24-well plates and each well contained 1.5]105 cells and 2 lMDCFH-DA in 2 ml medium. In the dose}response study (A), cellswere treated with different doses of silica for 4 h. In the time-course study (B), cells were treated with 100 lg/106 cells silica.Data are presented as means$SD (n"4); *P(0.05 compared tothe control group (one-way ANOVA with Scheffe’s test).

FIG. 3. The cytotoxicity of silica in AMs as evaluated by LDHleakage. In the dose}response study (A), cells were treated withdifferent doses of silica for 4 h. In the time-course study (B), cellswere treated with 100 lg silica/106 cells up to 4 h. Data are pre-sented as means$SD (n"4); *P(0.05 compared to the controlgroup (one-way ANOVA with Scheffe’s test).

248 ZHANG ET AL.

dose}response relationship when AMs were exposedto silica. It was found that LDH leakage in silica-treated AMs (100 lg/106 cells) reached about 80%after 4 h of treatment. The time-dependent changesof LDH leakage induced by silica are presented inFig. 3B. Signi7cant differences were observed from0.5 h onward. At the end of incubation, LDH leakagein silica-treated cells was six times higher than thecontrol.

Effect of Exposure to Silica on DNA Damage

The effects of silica on DNA damage were exam-ined using comet assay and the results are presentedin Fig. 4. There is a dose-dependent increase of DNAdamage in silica-treated AMs. It was also found thatAMs treated with 100 lg silica/106 cells for 4 h werein serious DNA damage, as most of the cells werefound to be in grades 3 and 4. No signi7cant DNA

damage was observed when cells were treated for1 and 2 h (data not shown).

Inhibitory Effects of Superoxide Dismutase andCatalase on Silica-Induced Oxidative Stress,Cytotoxicity, and Genotoxicity

The inhibitory effects of SOD and CAT againstsilica-induced oxidative stress, LDH leakage, andDNA damage are summarized in Fig. 5. In theseexperiments, AMs were pretreated with 250 U/mlSOD or 1000 U/ml CAT for 3 h prior to silica expo-sure. Figure 5A demonstrates that SOD displayedstrong scavenging capability against silica-inducedO~2 formation. The relative light unit of CL de-creased nearly 90% with the presence of SOD. CATwas also able to reduce the CL level, but to a lesserextent. Moreover, SOD and CAT were found to in-hibit DCF 8uorescence formation induced by silicawith similar capability (Fig. 5B). Both SOD and CAT

FIG. 4. Silica-induced DNA damage in AMs estimated withcomet assay. Alveolar macrophages were treated with differentconcentrations of silica for 4 h and the grading of cell damage wasestimated according to the amount of DNA in the comet tail undera 8uorescent microscope. Data are presented as percentage ofcells in each grade (n"4) and analyzed using the chi-square test.

SILICA-INDUCED OXIDATIVE STRESS 249

were capable of protecting against the cytotoxic ef-fect of silica. The percentages of LDH leakage weredecreased from 75 to 27% and 32% for SOD andCAT, respectively (Fig. 5C). The effects of SOD andCAT on silica-induced DNA damage are presented inFig. 5D. It was found that SOD and catalase exertedsigni7cant protective effects against silica-inducedDNA damage based on the following results: (1) a de-crease of the percentage of the cells in high grades(grades 3 and 4) and (2) on increase of the percentageof cells in grade 0.

DISCUSSION

Silica is a well-known occupational 7brogenicagent and its primary target cell is AMs. In thepresent study, we investigated the role of oxidativestress in silica-induced cytotoxicity and genotoxicityin cultured rat AMs. It was found that (1) silicaelevated the ROS level in AMs, as measured bylucigenin-dependent CL and DCFH-DA 8uores-cence; (2) silica is highly cytotoxic and genotoxic toAMs, as shown by LDH leakage and comet assay,respectively; and (3) silica-induced oxidative stressand toxicity were signi7cantly inhibited by two anti-oxidant enzymes (SOD and CAT). Therefore, resultsfrom this study provide strong evidence supportingthe notion that oxidative stress mediates thecytotoxicity and genotoxicity of silica in AMs.

Silica is known to be cytotoxic to the alveolarmacrophages, and the death of macrophages has

been suggested to be intimately related to thepathogenesis of silicotic 7brosis (Davis, 1986; Moss-man et al., 1998). The release of LDH, which indi-cates plasma membrane damage, has been widelyused to measure the cytotoxicity of various chem-icals both in vivo and in vitro (Grant et al., 1992).Our results indicate a potent cytotoxic effect of silica,as the percentage of LDH leakage reached about80% when AMs were treated with 100 lg silica/106

cells for 4 h, which was more than two times higherthan the effect of coal dust (30%) and TiO2 (27%),two relatively inert dusts. The initial interactionbetween silica particles and cell membrane is be-lieved to be through hydrogen bonding, or free-rad-ical-mediated reaction, or both (Erdogdu et al.,1998). It has been suggested that the cytotoxicity ofquartz is mainly due to the silanol groups that formon the surface of quartz particles, which might act assites for strong hydrogen bonding with the cell mem-brane (Shi et al., 1989). That might also explain thefact that silica-induced DNA damage observed inthis study is comparatively less extensive, as suchdamage is mainly mediated by ROS and the directcontact between silica and DNA is likely to be ratherlimited.

So far, evidence of silica-induced DNA damage ismainly based on either cell-free systems or in vivostudies. For instance, Shi et al. (1994, 1995) haveshown that silica causes DNA strand breaks in acell-free system. The involvement of ROS is stronglysuggested when the addition of 1.5% H2O2 to incuba-tions of DNA and silica greatly accelerated DNAstrand breakage (Daniel et al., 1995). Moreover, anin vivo study showed that the pulmonary 8-OHdGlevel increased more than twofold after exposure tosilica (Yamano et al., 1995). In our study, silica-induced DNA damage was assessed using comet as-say, a sensitive and reliable method for detectingDNA strand breaks at the individual cell level. Al-though it is true that DNA damage in AMs does notdirectly lead to carcinogenesis, as these cells do notreplicate themselves, it is still interesting to learnwhether silica exposure results in any DNA damagein AMs and about the involvement of ROS in suchdamage. It was found that silica treatment increasedDNA strand breaks in a dose-dependent manner(Fig. 4). Moreover, the two antioxidant enzymes(SOD and catalase) were able to inhibit silica-in-duced DNA damage (Fig. 5D). Therefore, the presentstudy provides direct evidence indicating the in-volvement of ROS in silica-induced DNA damage inAMs, its primary target cells. The role of ROS insilica-induced carcinogenesis has been suggested re-cently (Shi et al., 1998). Results from this study may

FIG. 5. The effects of SOD and CAT on silica-induced O~2 formation (A), H2O2 information (B), cytotoxicity (C), and genotoxicity (D) inAMs. Cells were pretreated with SOD (250 l/ml) or CAT (1000 l/ml) for 3 h prior to silica exposure (100 lg/106 cells). Data are presented asmeans$SD (n"4); *P(0.05 compared to the control group and dP(0.05 compared to silica group (one-way ANOVA with Scheffe’stest).

250 ZHANG ET AL.

help to further understand the mechanisms involvedin the carcinogenicity of silica.

Until recently, silica-induced oxidative stress andROS formation were mainly studied in the followingtwo aspects: (1) detection of Siz free radical formedon the surface of silica and the subsequent genera-tion of ROS upon the reaction with oxygen and aque-ous media (Shi et al., 1988, 1995; Dalal et al., 1990)and (2) the elevated ROS formation and up-regula-tion of antioxidant enzyme in lung tissue or cellsadministrated with silica (Vallyathan et al., 1995,1997). For instance, Dalal et al. (1990) demonstratedthe formation of silicon-based free radicals such asSiz, SiOz, and SiOOz on the surface of silica par-ticles. In a cell-free system, ESR spin-trappingmeasurements have shown that part of the oxygen

generated from silica particles can be trapped andO~2 is present on the surface of silica particles toreact with metal ions or reactive centers and toparticipate in chain reactions leading to more freeradical generation (Shi et al., 1995). For the majorityof these studies, the formation of ROS such as O~2 ,H2O2, zOH, and 1O2 was usually detected using ESRspin trapping, a special technique requiring sophis-ticated and expensive equipment. In the presentstudy, we used lucigenin-dependent CL for detectionof O~2 in silica-treated AMs. It is noted that theformation of O~2 occurred in less than 1 min after theaddition of silica, suggesting that silica induces theproduction of O~2 formation through the direct inter-action of silica particulate with plasma membraneprior to phagocytosis.

SILICA-INDUCED OXIDATIVE STRESS 251

In the present study, silica-induced oxidativestress in AMs was also studied using a DCFH-DA8uorescence test. Dichloro8uorescein 8uorescence isa sensitive, reliable indicator of intracellular H2O2

concentration (Bass et al., 1983; Lebel et al., 1992).The dose- and time-dependent increases of DCF 8uo-rescence indicated that silica treatment promotesthe production of H2O2. Indeed, an early study by Shiet al. (1988) revealed that freshly ground silica sus-pended in aqueous medium is able to generate H2O2.Subsequent studies of the mechanism involved insilica-induced zOH generation con7rm the formationof hydrogen peroxide from this silica reaction (Shiet al., 1998). Although H2O2 itself is not highly react-ive, its main mechanism is the formation of thehighly reactive species zOH via the metal-catalyzedHaber}Weiss reaction: O~2 #H2O2POH~#zOH#

H2O.Earlier studies using ESR showed that zOH gene-

ration induced by silica was inhibited in the pres-ence of SOD and CAT in aqueous suspension (Shiet al., 1989, 1994). Moreover, some in vivo experi-ments also demonstrated that silicotic lungs are ina state of oxidative stress and that increased genera-tion of ROS is associated with enhanced levels ofoxidative enzymes, including SOD, CAT, andglutathione peroxidase (Vallyathan et al., 1995; Val-lyathan and Shi, 1997). In the present study, wedemonstrated that SOD and CAT, two importantantioxidant enzymes, are able to protect against sil-ica-induced oxidative stress and cytotoxic andgenotoxic effects. Both enzymes may exert their pro-tective effects through the following pathways: (1)exogenous SOD and catalase scavenge ROS gene-rated extracellularly, either at the surface of cellmembranes or at the surface of silica particles; (2)both enzymes act intracellularly based on the evid-ence that they are able to enter AMs or other cellsthrough endocytosis (Beckman et al., 1988; Harrisonet al., 1994). The results from this study were foundto be consistent with a recent report showing thatlazaroid, a synthetic amino steroid with potent anti-oxidant activity, was able to protect againstsilica-induced cytotoxicity in rat AMs (Huang et al.,1998). It is well known that one of the importantfunctions of O~2 is to act as a reducing agent for Fe3`,which supplies Fe2` for the Haber}Weiss reaction tofacilitate zOH generation (Fridovich, 1986; Ito et al.,1992). Therefore, SOD not only directly scavengesO~2 , but also inhibits zOH formation indirectly.Catalase directly converts H2O2 into H2O and O2.The addition of SOD and CAT reduced the formationof Fe2` and H2O2, which then inhibited the produc-tion of zOH. Thus, the signi7cant inhibitory effects

of SOD and CAT clearly suggest that O~2 and H2O2

are the key intermediates in silica-induced cell injury.In summary, the present study systematically in-

vestigated the involvement of oxidative stress insilica-induced cytotoxicity and genotoxicity in AMs.The overall data provide convincing evidence thatoxidative stress mediates the silica-induced cyto-toxicity and genotoxicity. The understanding of sucha mechanism may provide a scienti7c basis for thepossible application of antioxidants in preventingthe hazardous effects of silica.

ACKNOWLEDGMENTS

The authors thank H. Y. Ong and Y. L. Chew for their technicalsupport. The present study was supported by the China MedicalBoard (New York), Program on Environmental and OccupationalHealth and the Center for Environmental and OccupationalHealth Research, National University of Singapore. The animalstudy in this project was conducted in accordance with nationaland institutional guidelines for the protection of animal welfare.

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