narayanan 1997

1997;57:3963-3971. Cancer Res P. K. Narayanan, E. H. Goodwin and B. E. Lehnert and Hydrogen Peroxide in Human Cells

Particles Initiate Biological Production of Superoxide Anionsα

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[CANCER RESEARCH57, 3963-3971, September 15, 1997J

production of ROS.3 Several lines of circumstantial evidence areconsistent with this possibility. For example, acute exposure to radonincreased SOD activity in the blood, kidney, liver, and spleen of rats(14), suggesting that a particles can up-regulate at least one antioxidant cellular defense mechanism in a directional manner to superoxideanions (O2@) and hydrogen peroxide (H2O2; Ref. 15). Longer exposures to radon, on the other hand, resulted in a decrease in SOD

activity in the same study (14), possibly because the stimulating effectin vivo may be a short-term phenomenon (14) or perhaps because ofan intracellular inactivation of SOD by ROS (16). More directly,Clutton and coworkers (17) have reported that the progeny of murinebone marrow cells that were exposed to high-linear energy transferneutrons showed an enhanced ability to oxidize intracellular DCFH,which was associated both with an increase in 8-hydroxy-2-deoxyguanine, an index of DNA oxidative base damage, and with DNAfragmentation. Further, we (18) recently found that exposure of serum-containing culture medium to a particles results in the generationof a short-lived SCE-inducing factor and that low dose a-irradiationof human cells results in the generation of a more persistent, heatlabile, cell-derived, SCE-inducing factor. These factors can induce

excessive SCEs in normal fibroblasts to the same extent as thatobserved when the cells are directly irradiated. In the context of ROS,both of the SCE-inducing responses to the factors were found to beinhibitable by SOD. Most simply, these latter observations are alsoconsistent with the possibility that a particles can damage DNAindirectly via a mechanism or mechanisms involving cellular gener

ation of ROS.Here, we investigated the hypothesis that a particles can induce the

cellular production of O2@ and H2O2 by assessing intracellular O2@and H2O2 with flow cytometry (19â€”21).We found that the intracellular production of O2@ and H202 is indeed increased followingexposure to low doses of a particles. Additionally, we report that suchincreases in intracellular ROS do not require direct nuclear or cellular/cytoplasmic hits by a particles and that the ROS response can be

induced by extracellular factors in a manner similar to which wepreviously observed with the induction of excessive SCEs (18).

MATERIALS AND METHODS

Reagents. Cells were harvested from culture plates using trypsin (0.25%)plus 1 mt@iEDTA (Life Technologies, Inc., Grand Island, NY). DCFH-DA

(Molecular Probes, Eugene, OR), dissolved in absolute ethanol (20 mM), wasused at a final concentration of 20 @xM.HE (Molecular Probes) was dissolved

in DMSO (Sigma Chemical Co., St. Louis, MO) to a concentration of 10 mM(final working concentration, 10 .LM).Hydrogen peroxide (H2O2; 30% w/v)was diluted in distilled water to a 20 mM stock solution and used at a final

concentration of 200 p@Mas a positive control because of its known capacity to

induce intracellular O2@ and H202 production in human cells (19). Sodium

azide (1 mM; Sigma) and diphenylenelodonium (50 jxM;Molecular Probes)were used to block mitochondrial and NADPH-oxidase stimulation, respectively (22). DMSO was used at a concentration of I % to scavenge hydroxylradicals (OH; Ref. 23).

3 The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dis

mutase; DCFH, 2',7'-dichlorofluorescin; SCE, sister chromatid exchange; DCFH-DA.DCFH diacetate; HE, hydroethidine; EB. ethidium bromide; DCF. 2',7'-dichlorofluorescein; DPI, diphenyleneiodonium.

3963

ABSTRACT

The mechanism(s) by which high-linear energy transfer a particles, likethose emitted by inhaled radon and radon daughters, cause lung cancerhas not been elucidated. Conceivably, DNA damage that is induced by aparticles may be mediated by the metabolic generation of reactive oxygenspecies (ROS), in addition to direct a particle.DNA interactions andhydroxyl radical-DNA interactions. Using normal human lung fibroblasts,we investigated the hypothesis that densely ionizing a particles mayInduce the intracellular generation of superoxide (Â°2@)and hydrogenperoxide (H2O@).Ethidium bromide and 2',7'-dichlorofluorescein, fluo.rescent products of the membrane-permeable dyes hydroethidine and2',7'-dichlorofluorescin diacetate, respectively, were used to monitor theintracellular production of O2@ and H202, respectively, by flow cytom.etry. Compared to sham.irradiated cells, fibroblasts that were exposed toa particles (0.4â€”19cGy) had significant Increases in intracellular Â°2production, along with concomitant increases in H202 production. Further analyses suggest that the plasma membrane.bound NADPH-oxidaseis primarily responsible for this increased intracellular generation of ROSand that the ROS response does not require direct nuclear or cellularâ€œhitsâ€•by the a particles. In this latter regard, we additionally report thatunirradiated cells also show the ROS response when they are incubatedwith serum-containing culture medium that has been exposed to a parti.des or when they are incubated with supernatants from a-irradiated cells.Our overall results support the possibility that a particles, at least in part,may mediate their DNA-damaging effects indirectly via a ROS-relatedmechanism.

INTRODUCTION

Epidemiological studies of uranium mine workers and experimentalanimal studies suggest a positive correlation between exposure to aparticles emitted from radon (222Rn) and its daughter products and thedevelopment of lung cancer (1â€”4).Although the mechanism(s) thatunderlie a particle-associated carcinogenesis remain unclear, wellrecognized cellular responses to a particles include chromosomeaberrations, gene mutations, induction of micronuclei, induction ofsister chromatid exchanges, cell cycle arrests, and lethality (5â€”10).Ithas been widely assumed that these effects, as well as the carcinogeniceffects of a particles, occur when a particles alter cellular DNA upondirect nuclear traversals. Indeed, numerous microdosimetric models,including the recently published Human Respiratory Tract Model forRadiological Protection, impose the assumption that a particle traversals of the nuclei of target cells in the mucosa lining the airways,e.g. , basal and secretory cells, are of primary concern for the induction

of lung cancer (11â€”13).Mounting evidence, however, indicates that a particles can cause

DNA damage by a mechanism or mechanisms that are independent ofnuclear traversals (7â€”9).Conceivably, the extranuclear effects of aparticles may, in some manner, be related to an a particle-associated

Received 4/4/97; accepted 7/18/97.Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page

charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicate this fact.

â€T̃his investigation was supported by United States Department of Energy-funded

project entifled â€œLowDose Ionizing Radiations, Reactive Oxygen Species, and GenomicInstability.â€•

2 To whom requests for reprints should be addressed. Phone: (505) 667-2753; Fax:

(505) 665-3024.

a Particles Initiate Biological Production of Superoxide Anions and Hydrogen

Peroxide in Human Cells1

P. K. Narayanan, E. H. Goodwin, and B. E. Lehne&

Cell and Molecular Biology Group, LS-4, Life Sciences Division, MS M888. Los Alamos National Laboratory. Los Alamos, New Mexico 87545




a PARTICLES AND ROS

Cell Culture. Normal human diploid lung fibroblasts (HFL1) obtainedfrom a human fetus (ATCC CCL 153; American Type Culture Collection,Rockville, MD) were cultured in 75-cm2 tissue culture flasks in a-MEM (Life

Technologies), supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT). All cell cultures were incubated at 37Â°Cin humidified5% CO2/95% air. Cells were harvested from the flasks by trypsinization andseeded into I .5-mm-thick, Mylar-bottomed, â€”30-mm-diameter culture dishes(24) at an initial density of 2 X l0@cells/dish 2 days prior to the exposures; allof the irradiations were performed on exponentially growing cells.

Exposure of Cells to a Particles. The HFL1 cells were exposed to dosesof a particles ranging from 0.4 to 19 cGy at room temperature using acollimated 238p.@a-particle exposure system that has been described in detailpreviously (25â€”27). With this system, the average energy of the a particles at

the cell-Mylar interface is â€”3.5MeV delivered at a dose rate of 3.65 cGysControl HFL1 cells were sham-irradiated at room temperature.

Assessment of Nuclear and Whole-Cell Hits and Extracellular Factorsas Inducers of Cellular ROS Production. Nuclear and cell morphometrywas performed to investigate whether detected increases in cellular ROS

production can occur in the absence of direct nuclear â€œhitsâ€•or even whole-cellhits by a particles. Exponentially growing HFL1 were fixed on the Mylar

dishes according to the protocol of Raju et al. (25), with minor modifications.

Briefly, the cells were first treated with 1% glutaraldehyde for 1 h at 4Â°C,followed by fixation in 100% methanol for 15 mm at â€”20Â°C.The cells were

then stained with 0. 1mg/mI Hoechst 33342 (Calbiochem-NovabiochemCorp.,La Jolla, CA) for 5 mm at 37Â°C,and the Mylar membranes were excised andmounted on glass slides using Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). Fields containing approximately 30â€”50cellswere viewed with a X20 Neofluor objective, the cells were imaged digitallyusing the image analysis software provided in the NIH Image version 1.54

program, run on a Macintosh Quadra 650 computer (28), and the mean nuclearand whole cell areas of the cells were determined (8). The values for nuclearand whole cell areas and target theory (29) were used in conjunction withinformation about a particle dose and fluence (8, 25) to calculate the mean

number of nuclear and whole cell hits at a given dose. The percentages of cellsthat were calculated to receive one or more nuclear and whole-cell hits by thea particles were then compared to the percentages of cells that were determined to have elevated ROS production by flow cytometry.

Assessment of a Particle-induced Extracellular Factors as Inducers ofCellular ROS Production. Supernatants harvested from cells immediately or24 h after exposure to 8.4- and l9-cGy doses of a particles were used inexperiments designed to assess a particle-induced extracellular factor(s) (18)as possible mediators of excessive ROS production. In these experiments, weparticularly focused on the intracellular production of H202. Cells that weresham-irradiated or that received culture medium from sham-irradiated cellsserved as negative controls. Cells exposed to 200 p@MH2O2served as routineinternal positive controls (20) for intracellular H202. In some experiments,various culture medium preparations in cell-free Mylar dishes were exposed toeither 8.4 or 19 cGy of a particles, or they were sham-irradiated prior to beingadded to otherwise untreated, exponential cultures of HFL1 . These latterexperiments were performed to determine whether or not a-irradiated culturemedium, which we previously have found to have SCE-inducing activity (18),can additionally stimulate the intracellular production of ROS.

In addition to investigating the H2O2-inducingcapacity of irradiated freshand conditioned medium, we also examined whether or not SOD (WorthingtonBiomedical Corp., Freehold, NJ), which inhibits the SCE-inducing activity of

irradiated medium (18), could also diminish the O2@ inducing potential ofa-irradiated medium. In these experiments, we pretreated the sham- or a-ir

radiated medium with 100 units/ml SOD before transfer onto unirradiatedHFL1.

Detection of Intracellular ROS. Intracellularmeasurementsof O2@usedHE, the sodium borohydride-reduced derivative of EB. During the oxidativeburst, stimulated fibroblasts produce O2@ via membrane-bound NADPH oxidase (30), resulting in an increase in red fluorescence (620 nm) due to the

conversion of HE to EB by O2@ . This can be measured directly by flow

cytometry (19). The specificity of HE for detecting O{ has been well

demonstrated in various cell types (19, 20).Intracellular H,O2 generation was detected using HFL1 loaded with

DCFH-DA (21). This assay involves the incorporation of DCFH-DA into thehydrophobic regions of the cell, where the acetate moieties are cleaved by

esterases, yielding the nonfluorescent molecule DCFH. DCFH is trapped dueto its polarity within the intracellular granules and the cytoplasm. The oxida

tive potentials of H202, together with peroxidases, are able to oxidize thetrapped DCFH to DCF, which is fluorescent in the green area of the spectrum,

i.e., 525 nm (19).

Prior to irradiation, the culture medium was replaced with HBSS supple

mented with 0.22% glucose, 2 mM glutamine, and 10% FCS (hereafter referredto as MHBSS). Unless otherwise indicated, all but residual MHBSS wasremoved from the Mylar dishes prior to the a- or sham-irradiations of cellcultures, and it was replaced immediately following exposure to prevent cell

dehydration. After irradiation, cells were removed from the Mylar using

trypsin-EDTA, and they were centrifuged and then resuspended in the MHBSSthat was present originally before irradiation. The ROS probes were thenadded, and the cells were incubated at 37Â°Cfor S mm. Thereafter, the tubeswere kept on ice. At this point, the positive control cells in unirradiatedMHBSS were treated with 200 @xMH2O2.All of the cell suspensions were thenfurther incubated for 30 mm at 37Â°C,put back on ice, and then immediatelyanalyzed on a flow cytometer. In some experiments, culture medium preparations in cell-free Mylar dishes were exposed to either 8.4 or 19 cOy of aparticles, or they were sham-irradiated prior to being added to unirradiated

cells that were preloaded with DCFH. After incubation for 30 mm or 3 h, thecells were examined by flow cytometry for DCF fluorescence. A similar

procedure was followed in experiments involving the cell-derived factor(s),except that the culture medium was left atop the monolayer of irradiated cellsfor 24 h before being transferred to unirradiated cells.

Flow Cytometry. The flow cytometric analyses were performed with a

Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, San Francisco, CA) using the instrument's standard computer, optics, and electronics. A15-mW, air-cooled, argon-ion laser was used as an excitation source for theROS-specific dyes at 488 nm. Optical filters placed in the fluorescencecollection pathway included a 530/30-nm band pass for DCF and a 585/42-nm

band pass for EB. Live HFL1 cells were distinguished from dead cells by using

measurements of forward angle light scattering and orthogonal light scatteringby virtue of their size and granularity of the cytoplasm (31). The cells werethen evaluated by applying a bitmap (gate) around the cell population withhigh light scattering signals, i.e. , viable cells. Fluorescent measurements were

collected from the cells in this gate and analyzed by the Cellquest data analysissoftware to assess the change in mean fluorescence between control and testsamples. The percentages of cells with increased signals were ascertained bysubtracting the number of events in each channel of the control histogram fromthat of the test histogram (32).

ROS Production m Cell-free Medium Only. We previously speculatedabout potential mechanisms that possibly could result in the generation of O2@and H2O2 upon exposure of serum-containing medium to a particles (18). Todetermine whether these ROS species are in fact generated in a-irradiated

medium, we performed cell-free assays to compare the oxidation of HE andDCFH in medium exposed to a particles, using potassium superoxide (KO{

Sigma, 0.0075â€”7.5 mM) and H2O2 (0.02â€”200 ,xM) as reference oxidants.Potassium superoxide at a concentration of 7.5 mM, equivalent to 200 p.MO2@,was used as a positive control for superoxide anions in this assay (33). For thedetection of O2, 10 ,.LMHE was added to HBSS with 0.22% glucose, 2 mM

glutamine, and 10% FCS, and medium samples in Mylar dishes were exposedto a particles at doses of 0.4, 3.6, and 19 cGy. Sham-irradiated medium

samples served as controls. Thereafter, the irradiated medium samples wereincubated at 37Â°Cfor 30 mm, followed by fluorescence measurements using a

Spex Fluorolog 1680double spectrofluorometer (Spex Industries Inc., Edison,NJ). Excitation was 488 nm with a 1-nmslit, and emission was collected at 610nm with a 1-nm slit. For the detection of H2O2 in the cell-free assay, thediacetate moiety of DCFH-DA was first cleaved by alkali treatment (0.2 N

NaOH; 1 mm at room temperature) to yield nonfluorescent DCFH, which issusceptible to H2O2-mediated oxidation (34). The pH of the resultant dye

solution was readjusted back to 7.4 before 20 nmi DCFH solution was addedto the medium. Following exposure to a particles at the above doses, thesamples were incubated at 37Â°Cfor 30 mm. After this incubation period,fluorescence emissions were collected at 5 14 nm with excitation of DCF at 488nm. Concentrations of O2@ and H202, produced as a result of exposure ofMHBSS to a particles (expressed as nanomolar), were then extrapolated fromthe standard curves via regression analysis.

3964




a PARTICLES AND ROS

r@@16@141.@

@ 12@1o

c.e@8

.E@6

@40@2

0e.0L) 15 30 45 60

Fig. 1. Time-dependent changes in intracellular O2@ production inHFL1 after sham-irradiation (0 cOy) and exposure to doses of aparticlesrangingfrom0.4 to 19cOy.Datapoints,means;bars, SE.Significantincreases(P < 0.05) in O2@production,measuredinterms of EB fluorescence, occurred in HFL1 at all time points following exposure to the a particles.

*

Statistical Analyses. Duplicate samples were run on the flow cytometer,and 5000 cells were analyzed from each tube. Data were expressed asmean Â±SE of replicate readings from a representative set of experiments andwere analyzed by one-way ANOVA followed by Tukey's procedure formultiple comparisons (35) using the VAX/VMS version of the Minitab statis

tical software package (Minitab, Inc., State College, PA). All flow cytometricand spectrofluorometric experiments were performed a minimum of six times,with the results from each being directionally reproducible. Ps of <0.05 wereconsidered to represent significant differences between group values.

RESULTS

Intracellular Superoxide and Hydrogen Peroxide Production in

Human Lung Fibroblasts Exposed to a Particles. Significant increases in mean EB fluorescence as an index of elevated O2@ production occurred relative to control values in all a-irradiated samples,as of the assay's first 15-mm time point (Fig. 1). These increases,however, did not show any obvious proportionality between dose andthe extent of HE oxidation to EB. Specifically, exposure to the8.4-cGy dose of a particles consistently resulted in the highest levelof O2@ production, whereas the lower and higher 0.4-, 3.6-, and

19-cGy doses resulted in lesser but similarly elevated levels of HEoxidation in repeated experiments. Over the remaining 45 mm of theassay, the oxidation of HE to EB appeared to progress linearly overtime, regardless of dose, at rates equivalent to that observed with thecontrol samples, i.e. , no significant differences among the slopes ofthe a- and sham-irradiated samples occurred between the 15- and60-mn time points (Fig. 1). These findings, accordingly, indicate that:exposure of normal human cells to a particles can induce the intracellular production of O2@ shortly after exposure, but enhancementsin the rates of O2@ production above basal levels cease within â€”15mm after exposure; and a particle-induced increases in O2@ do notconsistently scale with increasing exposure dose.

The patterns of change in mean intracellular DCF fluorescence asan index of intracellular H202 formation following exposure to the aparticles were generally much more complex than the previouslydescribed results obtained for O2@ production (Fig. 2). As seen earlierwith O2@ production, intracellular H2O2 generation showed significant increases, as of 15 mm following exposure to the 0.4â€”19-cOydoses of a particles. The increases in H2O2 generation after exposureto the higher doses (3.6, 8.4, and 19 cOy) peaked at 30 mm and then

IIJc)

30

25

20

15

10

5

Fig. 2. Time-dependent changes in intracellular H2O2production inHFLI after sham-irradiation (0 cOy) and exposure to a particle dosesranging from 0.4 to 19 cGy. Data points, means; bars, SE. Significant increases (P < 0.05) in H2O2production, measured in terms ofDCF fluorescence. occurred in HFL1 at all time points followingexposure to a particles.

*

**

*

*

â€”4â€”0cGy-@Iâ€”-O.4cGy-.4-- 1.5cGy--*[email protected]â€”4â€”19cGy

15 30 45 60TIME (minutes)

3965

* p < 0.05

TIME (minutes)

* p < 0.05




Table IPercentages of exponentially growing HFLI cells that received one or morenuclear or whole cell hits by aparticles%

of cells % of cells % changeinadosereceiving one receiving one or intracellularhydrogen(cGy)or

more nuclear hits more whole-cell hits peroxideproduction0.42.5

9.213.41.59.230.4493.620.858.2838.441.986.9971970.898.9 93

a PARTICLES AND ROS

0

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@ 800

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@40

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gradually decreased to levels that still remained well above controlvalues. On the other hand, the lower doses, 0.4 and 1.5 cGy, induceda gradual increase in intracellular H2O2 production that peaked at 45mm and remained elevated over unirradiated HFLI at 60 mm. Again,as found earlier with O2@ production, maximal increases in a partidc-induced H2O2 production did not scale with increasing exposuredose.

To identify the specific pathway responsible for this increase inH2O2 production, HFL1 cells were incubated with 1% DMSO, I mr@iazide, or 50 p@MDPI for 15 mm at 37Â°Cbefore exposure to a particles(Fig. 3). Pretreatment with DPI and DMSO substantially decreasedDCF fluorescence when pretreated cells were compared to untreated,a-irradiated cells. Pretreatment of cells with sodium azide, on theother hand, did not diminish the ROS response to a particle exposure.Because intracellular DCFH oxidation on exposure to a particles isinhibited by DPI and DMSO, the intracellular generation of ROSevidently is primarily the result of activating the NADPH oxidasecomplex that has been shown to exist in fibroblasts (30, 36, 37), andthe conversion of DCFH to fluorescent DCF involves the generationof OH radicals.

Relationships of Nuclear and Whole Cell Hits by a Particlesand the ROS Response. Table 1 summarizes the disproportionalitiesbetween the percentages of cells that produced intracellular H2O2 30mmaftera particleexposurerelativeto thepercentagesof cellsthatexperienced one or more nuclear or whole-cell hits by the a particles.The percentages of cells that showed higher intracellular H2O2 production measured in terms of change in mean intracellular DCFfluorescence were 5.4, 5.3, 4, 2.3, and 1.3 times higher than thepercentages ofcells that experienced nuclear hits at the 0.4-, 1.5-, 3.6-,8.4-, and l9-cGy doses, respectively. As well, the percentage of HFL1cells that showed higher H2O2 production were 1.5, 1.6, 1.4, and I .1

* p < 0.05

8.4 cGy (A)

Fig. 3. Intracellular H202 production in HFLI exposed to a particles(8.4 cOy) in the presence or absence of DMSO (OH radical scavenger.1%), sodium aside (mitochondrial inhibitor, 1 mM), and DPI (NADPHoxidase inhibitor, 50 jiM). Columns, means; bars, SE. H,O, productionwas markedly diminished in HFLI by preincubating the cells with 1%DMSOand 50 @.LMDPI for IS mmat 37Â°Cbeforeexposureto the aparticles. Azide did not alter a particle-induced ROS production inHFLI. *, significantly lower than values obtained with HFLI exposed toa particles alone (P < 0.05).

times higher than exponentially growing cells showing one or more

whole cell hits over the 0.4â€”8.4-cOy dose range. Thus, the intracellular generation of ROS in response to a-irradiation does not exclusively require direct nuclear or even whole-cell hits.

Origin of ROS-inducing Activity Present Immediately afterExposure to a Particles. We investigated the possibility that H2O2-inducing activity may be present in both a-irradiated fresh mediumand conditioned medium obtained from HFL1 exposed to a particlesin a manner akin to what we have observed previously in terms ofSCE-inducing activity (18). In these experiments, freshly preparedmedium samples (MEM + 10% fetal bovine serum) were irradiatedwith 0, 8.4, and 19 cGy of a particles. Immediately following theirradiations, the media were transferred onto unirradiated, exponentially growing recipient HFL1. The recipient cells were incubated withthe different medium samples for periods of 5 and 30 mm. The resultsfrom these experiments are summarized in Fig. 4. No significantdifferences were found between the mean DCF fluorescence in cellsreceiving sham- and a-irradiated fresh media at 5 mm (Fig. 4a).

However, at 30 mm, a significant difference was observed, with 23and 40% increases in H2O2 production at the 8.4- and l9-cGy doses,respectively (Fig. 4b). When unirradiated cells were exposed to a-irradiated conditioned medium, i.e. , medium taken from untreated cells,5 1 and 70% increases in DCF fluorescence were observed at 5 mm

with the 8.4- and l9-cGy irradiated medium samples, respectively(Fig. 5). Similar results were obtained with the recipient cells at the30-mm time point (Fig. 5). Consistent with the previous observationthat ROS production by cells does not require direct cell traversals bythe a particles, such findings indicate that ROS-inducing activity ispresent in cell-free culture medium shortly after exposure to a partides. Our findings, moreover, further suggest that ROS-inducing activity can arise from interactions between a particles and one or more

medium constituents, as well as from one or more constituents incell-conditioned medium.

Our previous study (18) revealed that exposing unirradiated HFL1cells to a-irradiated fresh or conditioned medium pretreated with SOD

reduced the occurrence of SCE to control levels. We therefore hypothesized that intracellular O2@ production in unirradiated HFL1exposed to a-irradiated (8.4 cGy) fresh and conditioned medium mayalso be inhibited by the addition of SOD. As summarized in Fig. 6,100 units/ml SOD reduced the O2@ production in unirradiated HFLIcells exposed to both a-irradiated fresh and conditioned medium by

3966

*

â€˜(A)+ 1% DMSO @(A)+5




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25 and 45%, respectively. These findings suggest that O2@ may heinvolved in the direct or indirect mediation of excessive SCE inresponse to a-irradiated medium.

Role of Cell.derived Factor(s) in ROS-inducing Activity afterExposure to a Particles. In addition to the role of an immediatefactor or factors in inducing SCEs in unirradiated HFLI, we previously found that a cell-derived SCE-inducing factor or factors werepresent in medium harvested from HFL1 cells 24 h after a-irradiation(18). In other analyses, the activity of the cell-derived factor(s) persisted even after freeze-thaw treatments and the medium was heated to56Â°Cfor 30 mm; only heating the medium to the boiling point ofwater (for 1 mm) destroyed the SCE-inducing activity (18). To determine whether a cell-derived factor or factors may cause a similarincrease in intracellular ROS production, exponentially growingHFL1 were a-irradiated in Mylar dishes at 0, 3.6, 8.4, and 19 cOy

after removal of culture medium. Medium was replaced on the directly irradiated cells, and the Mylar dishes put back into the incubatorfor 24 h (donors). Thereafter, the medium was then transferred ontoumrradiated exponentially growing HLF1 (recipients). Recipients

@ 20 _____

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@ 16

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a PARTICLES AND ROS

A B

* p < 0.05

Fig. 4. Intracellular H2O2 production in HFLI cells exposedto sham-irradiated (0 cGy) and a particle-irradiated fresh medium (8.4 and 19 cOy) at 5 (a) and 30 mm (b) after the onset ofincubation with the medium. Columns, means; bars, SE. *,significantly greater than with HFLI exposed to fresh mediumharvested from sham-irradiated HFLI.

8.4 19Alpha dose (cGy) Alpha dose (cGy)

were exposed to this medium for 5 mm, following which both donorsand recipients were harvested by trypsinization and collected in 15 ml

centrifuge tubes. Harvested cells were then centrifuged at 200 g for 5mm,and the cell pelletswereresuspendedin 500 pAof mediumcontaining DCFH-DA. The tubes were then incubated at 37Â°C for 5

mm for dye incorporation and then kept on ice. At this point, 200 p.MH,O2 was added to tubes set aside as positive controls, and all of thetubes were incubated simultaneously at 37Â°Cfor 30 mm. The cellsuspensions were then kept on ice and run subsequently on a flow

cytometer. Significant, dose-dependent increases in mean DCF fluorescence of recipient cells were seen at all doses, with the highest

increase occurring with cells that received the medium from HFL1

that had been irradiated with 19 cOy of a particles (Fig. 7). It is also

interesting to note that directly irradiated cells (donors) were stillcapable of producing significant amounts of H,O2 in a dose-dependent manner at 24 h (Fig. 7).

Cell-free Assay. A cell-free assay was performed to determine thepotential of a particles to oxidize HE to EB and DCFH to DCF,respectively, and to determine the equivalent amounts of O2 and

.5 mmâ€¢30mmn

â€”c-

*

*1

* p < 0.05

* *

-L-@

Fig. 5. Intracellular H2O2 production in HFLI cells at 5 and 30 mm afterincubation with sham-irradiated (0 cGy) and a particle-irradiated conditioned medium (8.4 and 19 cGy), i.e.. medium that was exposed to aparticles after removal from untreated, exponential HFLI . Columns,means; bars, SE. a, significantly higher than values obtained with HFLIexposed to conditioned medium harvested from sham-irradiated HFLI.

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3967

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8.4cGy@ 8.4 cOy+

SOD(100U)

gations have indicated that a particles can cause DNA alterations bya mechanism or mechanisms that are independent of nuclear or evenwhole-cell traversals (7, 8). Using the occurrence of excessive SCEsas an index of DNA damage in human lung fibroblasts, we (18)recently found that a relatively low dose of a particles results in thegeneration of extracellular factors, which, upon transfer to unexposed

normal human cells, can cause excessive SCE to an extent equivalentto that observed when the cells are directly irradiated with the sameirradiation dose. A short-lived, SCE-inducing factor or factors aregenerated in a-irradiated culture medium containing serum in the

absence of cells, and the activity of this factor or factors can bepromptly inhibited by SOD. A more persistent SCE-inducing factor orfactors, which can survive freeze-thawing, is heat labile and whichcan also be inhibited by SOD, was found to be produced by fibroblastsafter exposure to a particles. Such fmdings, in addition to other linesof suggestive evidence, prompted us to investigate the possibility that

Fig. 6. Intracellular O{ production in HFL1 exposed tosham-irradiated (0 cOy) and a-irradiated (8.4 cOy) fresh (a) orconditioned medium (b) in the presence or absence of Cu,ZnSOD (SOD)at 30 mm. UnirradiatedHFLI were incubatedwith sham- or a particle-irradiated medium, to which SOD wasadded immediately prior to transfer. Hydrogen peroxide (200lAM) was used as a positive control. Columns, means; bars, SE.

5, significantly different from HFL1 exposed to fresh or con

ditioned medium in the presence or absence of SOD harvestedfrom sham-irradiated HFLI.

*

r@:1

3968

* p < 0.05

*

shamirradiated

+SOD

(100U)

sham shamirradiated irradiated

+SOD

(100U)

H202 that may be produced in medium by the a particles. Significant,dose-dependent oxidations of HE to EB and DCFH to DCF wereobserved in a-irradiated MHBSS. The amounts of measured O2@produced by 0.4-, 3.6-, and 19-cGy a particles were â€”15,16, and 17flM, respectively (Table 2). The amounts of H2O2 detected were â€”7,

8, and 9 nM at the 0.4-, 3.6-, and 19-cOy exposure doses, respectively,or approximately 2-fold less than a particle-induced O2@ production.In comparison to these values, the concentrations of H2O2 produced ina similar system when HFLI were included along with the mediumwere â€”50â€”60times greater than levels produced in the cell-freesystem (Table 3).

DISCUSSION

Although direct nuclear traversals by a particles may be involved inmediating their mutagenic and carcinogenic effects, previous investi




a doseO2@production(cOy)(aM)

0.43.6

19

a dose (cOy)H,02 production(nM)0.4385

Â±IDâ€•3.6430Â±7â€•19562Â±

IIâ€•a

p < 0.05;significantlydifferentsham-irradiatedcontrol (a = 3).

a PARTICLES AND ROS

Fig. 7. Intracellular H2O2 production in recipient HFL1 cells cxposed to medium derived from HFLI exposed to 0, 8.4, and 19 cGyafter 24 h. Hydrogen peroxide production was monitored in recipientHFL1 for 30 mm and 3 h. Directly irradiated (donors) HFL1 cells werestill capable of producing significant amounts of H2O2 in a dosedependentmannerat 24 h. Columns,means;bars,SE. a, significantlydifferent from values obtained with HFL1 incubated in medium harvested from sham-irradiated HFL1.

DONORS (30 MIN) RECIPIENTS (30 MIN) DONORS (3 Lw) RECIPIENTS (3 Lw)

cOy, resulted in time-dependent increases in H2O2 production over a45-mnperiodbeforesubsiding,whereasH2O2valuesforthehigherdoses peaked either at 15 or 30 mm postexposure before showingdeclines. We presently do not have a firm, mechanistic explanation forthese complex H202 responses, but it is likely that they involvenumerous underlying and interrelating components that can contributeto the formation and removal of H2O2, e.g. , intracellular status ofSOD, iron, and catalase (38â€”40).Regardless, our collective resultssuggest that the a particle-induced ROS response involves mainly theplasma membrane-bound NADPH oxidase complex, in that the response was found to be inhibitable by diphenyleneiodonium, a selective inhibitor of the plasma membrane NADPH oxidase (41). Inaddition to implicating the generation of O{ as an inherent component of the response, this finding, along with results obtained with themitochondrial inhibitor sodium azide, shows that the increases inintracellular O2@ and H,O2 were not merely due to an enhanced stateof autoxidation of chemically reactive components produced duringreductive processes that are associated with the mitochondnal electrontransport system (42).

As with the induction of excessive SCE ( 18), the ROS responsedoes not require nuclear or whole-cell hits by the a particles for its

initiation. It can be induced by soluble, transmissible factors that aregenerated by the interaction of a particles with serum-containingculture medium or with cells. How the NADPH oxidase complexbecomes activated in response to exposure to a particles and thetransmissible factor(s) is unclear. We did observe that the a particleinduced oxidative burst in the fibroblasts could be inhibited byDMSO, a potent scavenger of OH radicals (23, 43). Although it iswell recognized that the presence of iron or other transitional metalcations can convert O2 and H,O, to @OHdownstream of activationof NADPH oxidase (44), the possibility that OH may initiate activation of NADPH oxidase, perhaps as a consequence of lipid peroxidation, cannot be ruled out (45). Nor can we rule out the possibility thatthe NADPH-oxidase complex is activated by O2@ or H2O2. Hydroxylradicals generated as a radiolytic product during the exposure of

Table 3 Hydrogen peroxide production measured on a spee:rofluorome:erfrom HFLJ(I X 1O@cells) exposedto a particles

Table 2 Superoxide and hydrogen peroxide production measured on aspectrofluorometer in a cell-free system

H2O2 production(aM)

14.9 Â±0.05â€• 6.9 Â±0.06a15.7 Â±o.o2a 8.2 Â±0.29Â°16.8Â±0.10Â° 8.7 Â±0.09Â°

a p < 0.05;significantlydifferentfromsham-irradiatedcontrols(n 3).

the extranuclear effects of a particles may be related to an a particleassociated production of ROS.

Here, we have presented new evidence that the intracellular production of O2' and H202 is indeed increased following exposure tolow doses of a particles. Moreover, our analyses indicate that theinduction of this ROS response does not require direct nuclear or evenwhole-cell hits by a particles. Furthermore, as in our previous investigation (18), in which SCE-inducing activity was found in cell-free

a-irradiated medium and in supernatants from a-irradiated cells, wehave shown that SOD-inhibitable, ROS-inducing activity is also present in such samples.

Our initial experiments involved measuring the intracellular ROSinducing activity of a particles in terms of post exposure time anddose. Even at the lowest dose studied (0.4 cOy), intracellular O2@significantly increased in cells shortly after exposure to the a partides. Enhancements in the rates of O2@ production above basal levels,however, were short-lived, i.e. , they ceased within â€”15 mm afterexposure. Interestingly, the a particle-induced increases in O2@ didnot consistently scale with increasing exposure dose, but, instead, theinduction of O2@ seemingly occurred in an â€œall-or-noneâ€•fashionwithin the dose range we examined. Of possible mechanistic relevance, a virtual constancy in the mean number of induced SCEs/cell,independent of the dose of a-particles administered, was also observed previously, once a dose threshold was reached (8).

The intracellular production of H2O2 as a function of dose andpostexposure time, on the other hand, was much more complex. Someevidence for a dose-response relationship was observed as of 15 mmafter exposure over the 0.4â€”3.6-cOy dose range. However, such arelationship did not continue with the higher 8.4- and 19-cOy doses,

which actually resulted in lesser amounts of H2O2 at the 15 mmpostexposure time point than what was observed with the lower3.6-cOy dose. In terms of the postexposure kinetic patterns of H2O2production, exposure to the lower doses of a particles, i.e. , 0.4 and 1.5

3969




a PARTICLESANDROS

ROS in cells from individuals with the cancer-prone disorder Bloom'ssyndrome (55, 63). Conceivably, the induction of SCE by a particlesmay at least in part reflect an alteration a cell's state of oxidantantioxidant imbalance. With regard to carcinogenesis, substantial cvidence exists in support of ROS as mediators of radiation-induceddamage in a variety of systems (64). ROS cause formation of oxidizedbases and a spectrum of DNA lesions including base damage, singlestrand breaks, double-strand breaks, cross-linking of DNA, and damage to the deoxyribose moiety (64â€”70). Other lines of evidenceindicate that ROS play important roles in virtually all stages oftumorigenesis initiated by ionizing radiation (65). McLennan et a!.(70) demonstrated some time ago that OH radicals generated from02@ H2O2, and iron were the agents responsible for oxygen-inducedcellular damage and death (70), and more recent studies have shownthat exposure of eukaryotic cells to radiation is associated with induction of oxidative stress and attendant DNA damage, inter a!ia (48).Extension of our present findings to the in vivo condition wouldsuggest that inhaled radon and radon progeny may induce a conditionof oxidative stress that is transmissible among lung cells and that maybe involved in mediating DNA damage.

ACKNOWLEDGMENTS

We express our gratitude to A. Deshpande, Y. Valdez, and Y. Shou for theirtechnical assistance during the course of this study.

REFERENCES

1. Samet, J. M. Radon and lung cancer. J. Natl. Cancer Inst. (Bethesda), 81: 745â€”757,1989.

2. Lubin, J. H., and Boice, J. D. Lung-cancer risk from residential radon: metaanalysisof 8 epidemiologic studies. J. Nail. Cancer Inst. (Bethesda), 89: 49â€”57,1997.

3. Lubin, J. H., Boice, J. D., Jr., and Edling, C. Radon and Lung Cancer Risk: A JointAnalysis of II Underground Miner Studies. Bethesda: NIH, 1994.

4. Puskin, J. S., and Boice, J. D., Jr. EPA's perspective on risks from residential radonexposure. J. Air Pollut. Control Assoc., 39: 915â€”920,1989.

5. Kennedy, C. H., Mitchell, C. E., Fukushima, N. H., Neft, R. E., and Lechner, J. F.Induction of genomic instability in normal human bronchial epithelial cells by 238p.,ja-particles. Carcinogenesis (Lond.), 17: 1671â€”1676,1996.

6. Brooks, A. L., Newton, G. J., Shyr, L-J., Seiler, F. A., and Scott, B. R. The combinedeffects of a-particles and X-rays on cell killing and micronuclei induction in lungepithelial cells. Int. J. Radiat. Biol., 58: 799â€”811, 1990.

7. Nagasawa, H., Little, J. B., Inkret, W. C., Carpenter, S., Raju, M. R., Chen, D. J., andStrniste, G. F. Response of X-ray sensitive CHO mutant cells (xrs-6c) to radiation. II.Relationship between cell survival and the induction of chromosomal damage withlow doses of a particles. Radiat. Res., 126: 280â€”288,1991.

8. Deshpande, A., Goodwin, E. H., Bailey, S. M., Marrone, B. L., and Lehnert, B. E.a-particle-induced sister chromatid exchange in normal human lung fibroblasts:evidence for an extranuclear target. Radiat. Res., 145: 260â€”267, 1996.

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I 1. Intemational Council for Radiation Protection. Human respiratory tract model forradiological protection. in: Annals of the ICRP24. ICRP Publ. No. 66., pp. 1â€”3.London: Pergamon Press, 1994.

12. James, A. C. Lung dosimetry. In: A. V. Nero (ed), Radon and Its Decay Products inIndoor Air, pp. 259â€”309.New York: John Wiley & Sons, Inc., 1988.

13. Hui, T. E., Poston, J. W., and Fisher, D. R. The microdosimetry of radon decayproducts in the respiratory tract. Radiat. Prot. Dosim., 31: 405â€”411, 1990.

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15. Spolarics, Z. Endotoxin stimulates gene-expression of ROS-eliminating pathwaysin rat hepatic endothelial and kupffer cells. Am. J. Physiol., 33: G660â€”0666,1996.

16. lizawa, 0., Kato, T., Tagami, H., Akamatsu, H., and Niwa, Y. Long-term follow-upstudy of changes in lipid peroxide levels and the activity of superoxide dismutase,catalase, and glutathione peroxidase in mouse skin after acute and chronic UVirradiation. Arch. Dermatol. Res., 286: 47â€”52,1994.

17. Clutton, S. M., Townsend, K. M. S., Walker, C., Ansell, J. D., and Wright, E. G.Radiation-induced genomic instability and persisting oxidative stress in primarybone-marrow cultures. Carcinogenesis (Land.), 17: 1633â€”1639,1996.

18. Lehnert, B. E., and Goodwin, E. H. Extracellular factor(s) following exposure to aparticles can cause sister chromatid exchanges in normal human cells. Cancer Rca.,57: 2164â€”2171,1997.

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3970

medium to a particles would be expected to be short-lived, with a z@of â€”l0@ 5 (46); no transmissible ROS-inducing activity from thissource of @OHwould be expected. It may be possible, however, for0H to be produced in the medium in a more sustained manner. Forexample, one mechanism for a more persistent production of OHcould begin with the generation of O2@, which can arise from theinteraction of molecular oxygen with either electrons that are ejectedfrom water by ionizing radiation or with the radiolytic product if(Caq + 02@ O2@; if + 02 @â‚¬O2@ + H@; Ref. 47). Conversion to

@OHcould then proceed via an iron-catalyzed Haber-Weiss reaction,i.e., O2@ + H2O2 iâ‚¬02 + OH + OH (48), after dismutation ofsome O2@ to H2O2, which, expectedly, would be facilitated byextracellular SOD that was present in serum (49â€”51). Hydroxylradicals from this source, in addition to 0H initially produced radiolytically, in turn could form a second source for the further productionof O2@ and, subsequently, the further production of OH. Accordingto this scheme,@OHradicalsabstracthydrogenatomsfrom thecarbonchains of unsaturated fatty acids present in serum or cell membranesto yield peroxy radicals. Further hydrogen abstraction by the peroxyradicals results in the formation of lipid peroxides, which also caninitiate a chain reaction leading to peroxidation of a large number ofunsaturated fatty acids. It is well established that lipid peroxidationcan simultaneously lead to the generation of numerous metabolitesthat have the propensity to induce O2@ generation (52â€”54).Further@OHgeneration would then follow, as described before, with repetitionof these reactions occurring until they were limited by substrateavailability. Consistent with these possibilities, spectrofluorometricanalyses of a-irradiated cell-free medium containing DCFH-DA andHE did reveal that exposure of medium to a particles resulted in thedetectable generation of putative O2@ and H2O2. A central role forO2' in the process of NADPH oxidase activation, at least, is underscored by our finding that the ROS-inducing activity in irradiatedmedium can be diminished by SOD.

A second source of ROS-inducing factor(s) was also found to bepresent in cell supernatants harvested up to 24 h after the cells were

a-irradiated. In follow-up experiments, we have found that the productionof this factor or factors can be inhibited by cycloheximide treatment ofthe a-irradiated cell cultures (data not shown), which suggests that thecell-derived factor(s) may be proteinaceous in nature. How this factor orfactors mediate their ROS-inducing effect remains to be elucidated, but itis noteworthy that several cell-derived factors have now been identifiedthat can cause DNA damage via as yet poorly understood mechanisms.Conditioned media from cultures of cells from subjects with the cancerpronedisordersBloom's syndrome,Fanconi'sanemia,andataxia-telangiectasia, for example, cause chromosomal aberrations and increasedincidence of SCE in normal cells (55â€”57).Clastogenic activity also hasbeen observed in the plasma of individuals that received very high, wholebody doses of low-linear energy transfer ionizing radiation and after

therapeutic exposure to X-rays (58â€”60).Such activity additionally hasbeen found in plasma that was irradiated in vitro (61). A variety ofchemical species have been proposed to function as DNA-damagingfactors, e.g., aldehyde breakdown products of lipid peroxidation, tumornecrosis factor-a, and inosine nucleotides (55, 58, 62), but aside fromhaving a Mr of <10,000 and oftentimes being inhibitable by antioxidants(55), the exact identification of such factors at play in the above conditions as well as in response to exposure to a particles remains to bedetermined.

It is tempting to speculate about potential mechanistic links between a particle-induced SCE, the intracellular generation of ROS,and a particle-induced carcinogenesis. Unfortunately, possible associations among these outcomes in terms of cause-and-effect relationships are at most currently circumstantial. As examples, excessiveSCEs have been associated with an increased state of production of




a PARTICLES AND RO5

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