Particles Initiate Biological Production of Superoxide Anions and Hydrogen Peroxide in Human Cells

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

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

a Particles

Initiate

Biological

Production

of Superoxide

Anions

and Hydrogen

Peroxide

in Human

Cells1
and B. E. Lehne&

P. K. Narayanan,

E. H. Goodwin,

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

ABSTRACT The mechanism(s) by which high-linear energy transfer a particles, like

those emitted by inhaled radon and radon daughters, cause lung cancer

has not been elucidated. Conceivably, DNA damage that is induced by a particles may be mediated by the metabolic generation of reactive oxygen
species (ROS), in addition to direct a particle.DNA interactions and

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

hydroxyl radical-DNA interactions. Using normal human lung fibroblasts, we investigated the hypothesis that densely ionizing a particles may Induce the intracellular generation of superoxide (2@)and hydrogen peroxide (H2O@).Ethidium bromide and 2',7'-dichlorofluorescein, fluo. rescent products of the membrane-permeable dyes hydroethidine and 2',7'-dichlorofluorescin diacetate, respectively, were used to monitor the intracellular production of O2@ and H202, respectively, by flow cytom. etry. Compared to sham.irradiated cells, fibroblasts that were exposed to a particles (0.419 cGy) had significant Increases in intracellular 2 production, along with concomitant increases in H202 production. Fur ther analyses suggest that the plasma membrane.bound NADPH-oxidase
is primarily responsible for this increased intracellular generation of ROS

and that the ROS response does not require direct nuclear or cellular hits by the a particles. In this latter regard, we additionally report that
unirradiated cells also show the ROS response when they are incubated

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

with 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-related mechanism. INTRODUCTION Epidemiological studies of uranium mine workers and experimental animal studies suggest a positive correlation between exposure to a particles emitted from radon (222Rn) and its daughter products and the development of lung cancer (14).Although the mechanism(s) that underlie a particle-associated carcinogenesis remain unclear, well recognized cellular responses to a particles include chromosome aberrations, gene mutations, induction of micronuclei, induction of sister chromatid exchanges, cell cycle arrests, and lethality (510). It has been widely assumed that these effects, as well as the carcinogenic effects of a particles, occur when a particles alter cellular DNA upon direct nuclear traversals. Indeed, numerous microdosimetric models, including the recently published Human Respiratory Tract Model for Radiological Protection, impose the assumption that a particle tra versals 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

excessive SCEs in normal fibroblasts to the same extent as that observed when the cells are directly irradiated. In the context of ROS, both of the SCE-inducing responses to the factors were found to be inhibitable by SOD. Most simply, these latter observations are also consistent with the possibility that a particles can damage DNA
indirectly 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 (1921).We found that the intracel lular production of O2@ and H202 is indeed increased following exposure to low doses of a particles. Additionally, we report that such increases 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 we previously 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), was
used at a final concentration of 20 @xM. HE (Molecular Probes) was dissolved

of lung cancer (1113). Mounting evidence, however, indicates that a particles can cause DNA damage by a mechanism or mechanisms that are independent of nuclear traversals (79).Conceivably, the extranuclear effects of a particles may, in some manner, be related to an a particle-associated
Received 4/4/97; accepted 7/18/97.

in DMSO (Sigma Chemical Co., St. Louis, MO) to a concentration of 10 mM

(final working concentration,
was diluted concentration in distilled

10 .LM).Hydrogen peroxide (H2O2; 30% w/v)

and used at a final to control because of its known capacity

water to a 20 mM stock solution

of 200 p@M as a positive

induce intracellular

O2@ and H202 production

in human cells (19). Sodium

azide (1 mM; Sigma) and diphenylenelodonium (50 jxM;Molecular Probes) Thecostsof publication of thisarticleweredefrayedin partby the paymentof page charges.Thisarticlemustthereforebe herebymarkedadvertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
This investigation was supported by United States Department of Energy-funded

were used to block mitochondrial and NADPH-oxidase stimulation, respec tively (22). DMSO was used at a concentration of I % to scavenge hydroxyl radicals (OH; Ref. 23).
3 The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dis

project entifled Low Dose Ionizing Radiations, Reactive Oxygen Species, and Genomic
Instability.
2 To whom requests for reprints should be addressed. Phone: (505) 667-2753; Fax:

(505) 665-3024.

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

3963

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a PARTICLES

AND ROS

Cell Culture. Normal human diploid lung fibroblasts (HFL1) obtained

from 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 Labora

esterases, yielding the nonfluorescent molecule DCFH. DCFH is trapped due to its polarity within the intracellular granules and the cytoplasm. The oxida tive potentials of H202, together with peroxidases, are able to oxidize the trapped 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

tories, Inc., Logan, UT). All cell cultures were incubated at 37C in humidified 5% CO2/95% air. Cells were harvested from the flasks by trypsinization and
seeded into I .5-mm-thick, Mylar-bottomed, 30-mm-diameter culture dishes (24) at an initial density of 2 X l0@cells/dish 2 days prior to the exposures; all of the irradiations were performed on exponentially growing cells.

mented with 0.22% glucose, 2 mM glutamine, and 10% FCS (hereafter referred

to as MHBSS). Unless otherwise indicated, all but residual MHBSS was removed from the Mylar dishes prior to the a- or sham-irradiations of cell
cultures, and it was replaced After irradiation, immediately cells were following removed exposure from to prevent the Mylar cell using dehydration.

Exposure of Cells to a Particles. The HFL1 cells were exposed to doses

of a particles ranging from 0.4 to 19 cGy at room temperature using a collimated 238p.@ a-particle exposure system that has been described in detail
previously (2527). With this system, the average energy of the a particles at

trypsin-EDTA, and they were centrifuged and then resuspended in the MHBSS
that was present originally before irradiation. The ROS probes were then added, and the cells were incubated at 37Cfor S mm. Thereafter, the tubes were kept on ice. At this point, the positive control cells in unirradiated

the cell-Mylar interface is 3.5 MeV delivered at a dose rate of 3.65 cGys Control HFL1 cells were sham-irradiated at room temperature. Assessment of Nuclear and Whole-Cell Hits and Extracellular Factors
as Inducers of Cellular ROS Production.
whether

Nuclear and cell morphometry

increases were in cellular ROS

was performed

to investigate Exponentially

detected HFL1

rations in cell-free Mylar dishes were exposed to either 8.4 or 19 cOy of a particles, or they were sham-irradiated prior to being added to unirradiated dishes according to the protocol of Raju et al. (25), with minor modifications. cells that were preloaded with DCFH. After incubation for 30 mm or 3 h, the Briefly, the cells were first treated with 1% glutaraldehyde for 1 h at 4C, cells were examined by flow cytometry for DCF fluorescence. A similar followed by fixation in 100% methanol for 15 mm at 20C. The cells were procedure was followed in experiments involving the cell-derived factor(s), then stained with 0. 1 mg/mI Hoechst 33342 (Calbiochem-Novabiochem Corp., except that the culture medium was left atop the monolayer of irradiated cells La Jolla, CA) for 5 mm at 37C,and the Mylar membranes were excised and for 24 h before being transferred to unirradiated cells. mounted on glass slides using Vectashield mounting medium (Vector Labo Flow Cytometry. The flow cytometric analyses were performed with a ratories, Inc., Burlingame, CA). Fields containing approximately 3050cells Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, San Fran were viewed with a X20 Neofluor objective, the cells were imaged digitally cisco, CA) using the instrument's standard computer, optics, and electronics. A
hits by a particles. growing fixed on the Mylar using the image analysis software provided in the NIH Image version 1.54

production can occur in the absence of direct nuclear hits or even whole-cell

MHBSS were treated with 200 @xM H2O2.All of the cell suspensions were then further incubated for 30 mm at 37C, put back on ice, and then immediately analyzed on a flow cytometer. In some experiments, culture medium prepa

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

15-mW, air-cooled, argon-ion laser was used as an excitation source for the ROS-specific dyes at 488 nm. Optical filters placed in the fluorescence
collection pathway included a 530/30-nm band pass for DCF and a 585/42-nm

with

the mean

number of nuclear and whole cell hits at a given dose. The percentages of cells that were calculated to receive one or more nuclear and whole-cell hits by the

band pass for EB. Live HFL1 cells were distinguished from dead cells by using measurements of forward angle light scattering and orthogonal light scattering

by virtue of their size and granularity of the cytoplasm (31). The cells were
then evaluated by applying a bitmap (gate) around the cell population with high light scattering signals, i.e. , viable cells. Fluorescent measurements were

a particles were then compared to the percentages of cells that were deter
mined to have elevated ROS production by flow cytometry.

Assessment of a Particle-induced Extracellular Factors as Inducers of

Cellular ROS Production. Supernatants harvested from cells immediately or

24 h after exposure to 8.4- and l9-cGy doses of a particles were used in

experiments designed to assess a particle-induced extracellular factor(s) (18) as possible mediators of excessive ROS production. In these experiments, we

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

particularly focused on the intracellular production of H202. Cells that were sham-irradiated or that received culture medium from sham-irradiated cells served as negative controls. Cells exposed to 200 p@M H2O2served as routine
internal positive controls (20) for intracellular H202. In some experiments,

ROS Production m Cell-free Medium Only. We previously speculated

about potential mechanisms that possibly could result in the generation of O2@ and H2O2 upon exposure of serum-containing medium to a particles (18). To
determine whether these ROS species are in fact generated in a-irradiated

various culture medium preparations in cell-free Mylar dishes were exposed to

either 8.4 or 19 cGy of a particles, or they were sham-irradiated prior to being added to otherwise untreated, exponential cultures of HFL1 . These latter

medium, we performed cell-free assays to compare the oxidation of HE and

DCFH in medium exposed to a particles, using potassium superoxide (KO{ Sigma, 0.00757.5 mM) and H2O2 (0.02200 ,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 the detection of O2, 10 ,.LMHE was added to HBSS with 0.22% glucose, 2 mM

experiments were performed to determine whether or not a-irradiated culture

medium, which we previously have found to have SCE-inducing
can additionally stimulate the intracellular production of ROS.

activity (18),

In addition to investigating the H2O2-inducingcapacity of irradiated fresh

and conditioned medium, we also examined whether or not SOD (Worthington
Biomedical a-irradiated Corp., Freehold, medium. NJ), which inhibits the SCE-inducing we pretreated activity of or a-ir

glutamine, and 10% FCS, and medium samples in Mylar dishes were exposed
to a particles at doses of 0.4, 3.6, and 19 cGy. Sham-irradiated medium samples served as controls. Thereafter, the irradiated medium samples were incubated at 37Cfor 30 mm, followed by fluorescence measurements using a

irradiated medium (18), could also diminish the O2@ inducing potential of
In these experiments, the sham-

radiated medium with 100 units/ml SOD before transfer onto unirradiated HFL1.

Spex Fluorolog 1680 double spectrofluorometer (Spex Industries Inc., Edison, NJ). Excitation was 488 nm with a 1-nmslit, and emission was collected at 610
nm with a 1-nm slit. For the detection of H2O2 in the cell-free assay, the diacetate moiety of DCFH-DA was first cleaved by alkali treatment (0.2 N NaOH; 1 mm at room temperature) to yield nonfluorescent DCFH, which is susceptible to H2O2-mediated oxidation (34). The pH of the resultant dye solution was readjusted back to 7.4 before 20 nmi DCFH solution was added to the medium. Following exposure to a particles at the above doses, the samples were incubated at 37Cfor 30 mm. After this incubation period, fluorescence emissions were collected at 5 14 nm with excitation of DCF at 488

Detection of Intracellular ROS. Intracellular measurements of O2@used HE, the sodium borohydride-reduced derivative of EB. During the oxidative burst, stimulated fibroblasts produce O2@ via membrane-bound NADPH ox
idase (30), resulting in an increase in red fluorescence (620 nm) due to the conversion cytometry of HE to EB by O2@ . This can be measured directly by flow (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 the
hydrophobic regions of the cell, where the acetate moieties are cleaved by

nm. Concentrations of O2@ and H202, produced as a result of exposure of MHBSS to a particles (expressed as nanomolar), were then extrapolated from
the standard curves via regression analysis.

3964

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a PARTICLES

AND ROS

r@
@16@141.@

Fig. 1. Time-dependent

changes in intracellular O2@ production in

12@1o
c.e@8
*

HFL1 after sham-irradiation (0 cOy) and exposure to doses of a

particlesrangingfrom0.4 to 19cOy.Datapoints,means;bars, SE. Significantincreases(P < 0.05) in O2@production,measuredin

terms of EB fluorescence, occurred in HFL1 at all time points follow ing exposure to the a particles.

* p < 0.05

.E@6
@40@2
0 .0

L)

15

30
TIME (minutes)

45

60

Statistical

Analyses.

Duplicate samples were run on the flow cytometer,

and 5000 cells were analyzed from each tube. Data were expressed as
mean SE of replicate readings from a representative set of experiments and were analyzed by one-way ANOVA followed by Tukey's procedure for
multiple comparisons (35) using the VAX/VMS version of the Minitab statis

tical software package (Minitab, Inc., State College, PA). All flow cytometric

and spectrofluorometric experiments were performed a minimum of six times, with the results from each being directionally reproducible. Ps of <0.05 were considered to represent significant differences between group values.

RESULTS
Intracellular Superoxide and Hydrogen Peroxide Production in

Human Lung Fibroblasts Exposed to a Particles. Significant in creases in mean EB fluorescence as an index of elevated O2@ pro duction 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 and the extent of HE oxidation to EB. Specifically, exposure to the 8.4-cGy dose of a particles consistently resulted in the highest level of O2@ production, whereas the lower and higher 0.4-, 3.6-, and

19-cGy doses resulted in lesser but similarly elevated levels of HE oxidation in repeated experiments. Over the remaining 45 mm of the assay, the oxidation of HE to EB appeared to progress linearly over time, regardless of dose, at rates equivalent to that observed with the control samples, i.e. , no significant differences among the slopes of the a- and sham-irradiated samples occurred between the 15- and 60-mn time points (Fig. 1). These findings, accordingly, indicate that: exposure of normal human cells to a particles can induce the intra cellular production of O2@ shortly after exposure, but enhancements in the rates of O2@ production above basal levels cease within 15 mm after exposure; and a particle-induced increases in O2@ do not consistently scale with increasing exposure dose. The patterns of change in mean intracellular DCF fluorescence as an index of intracellular H202 formation following exposure to the a particles were generally much more complex than the previously described results obtained for O2@ production (Fig. 2). As seen earlier with O2@ production, intracellular H2O2 generation showed signifi cant increases, as of 15 mm following exposure to the 0.419-cOy doses of a particles. The increases in H2O2 generation after exposure to the higher doses (3.6, 8.4, and 19 cOy) peaked at 30 mm and then

30
*

25
* *

20
*

I
c)

15 10
5

Fig. 2. Time-dependent changes in intracellular H2O2production in HFLI after sham-irradiation (0 cOy) and exposure to a particle doses ranging from 0.4 to 19 cGy. Data points, means; bars, SE. Signifi cant increases (P < 0.05) in H2O2production, measured in terms of DCF fluorescence. occurred in HFL1 at all time points following
exposure to a particles. * p < 0.05

40cGy -.4--

-@I-O.4 cGy
1.5cGy

--*--3.6cGy -@I--8.4 cGy 419 cGy

15

30

45
3965

60

TIME (minutes)

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a PARTICLES AND ROS

1@ C

120

* p < 0.05

F00
@
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 (NADPH oxidase inhibitor, 50 jiM). Columns, means; bars, SE. H,O, production 0

80

was markedly diminished in HFLI by preincubating the cells with 1%

DMSOand 50 @.LM DPI for IS mm at 37C beforeexposureto the a

particles. Azide did not alter a particle-induced ROS production in HFLI. *, significantly lower than values obtained with HFLI exposed to a particles alone (P < 0.05). 0
*

@40
C C

8.4 cGy (A)

(A) + 1% DMSO

@(A) +5

gradually decreased to levels that still remained well above control values. On the other hand, the lower doses, 0.4 and 1.5 cGy, induced a gradual increase in intracellular H2O2 production that peaked at 45 mm and remained elevated over unirradiated HFLI at 60 mm. Again, as found earlier with O2@ production, maximal increases in a parti dc-induced H2O2 production did not scale with increasing exposure dose. To identify the specific pathway responsible for this increase in H2O2 production, HFL1 cells were incubated with 1% DMSO, I mr@i azide, or 50 p@M DPI for 15 mm at 37C before exposure to a particles (Fig. 3). Pretreatment with DPI and DMSO substantially decreased DCF fluorescence when pretreated cells were compared to untreated, a-irradiated cells. Pretreatment of cells with sodium azide, on the other hand, did not diminish the ROS response to a particle exposure. Because intracellular DCFH oxidation on exposure to a particles is inhibited by DPI and DMSO, the intracellular generation of ROS evidently is primarily the result of activating the NADPH oxidase complex that has been shown to exist in fibroblasts (30, 36, 37), and the conversion of DCFH to fluorescent DCF involves the generation of OH radicals. Relationships of Nuclear and Whole Cell Hits by a Particles and the ROS Response. Table 1 summarizes the disproportionalities between the percentages of cells that produced intracellular H2O2 30

times

higher

than exponentially

growing

cells showing

one or more

whole cell hits over the 0.48.4-cOy dose range. Thus, the intracel lular generation of ROS in response to a-irradiation does not exclu sively require direct nuclear or even whole-cell hits. Origin of ROS-inducing Activity Present Immediately after Exposure to a Particles. We investigated the possibility that H2O2inducing activity may be present in both a-irradiated fresh medium and conditioned medium obtained from HFL1 exposed to a particles in a manner akin to what we have observed previously in terms of SCE-inducing activity (18). In these experiments, freshly prepared medium samples (MEM + 10% fetal bovine serum) were irradiated with 0, 8.4, and 19 cGy of a particles. Immediately following the irradiations, the media were transferred onto unirradiated, exponen tially growing recipient HFL1. The recipient cells were incubated with the different medium samples for periods of 5 and 30 mm. The results from these experiments are summarized in Fig. 4. No significant differences were found between the mean DCF fluorescence in cells
receiving shamand a-irradiated fresh media at 5 mm (Fig. 4a).

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

mmaftera particle exposure relative to thepercentages of cellsthat

experienced one or more nuclear or whole-cell hits by the a particles. The percentages of cells that showed higher intracellular H2O2 pro duction measured in terms of change in mean intracellular DCF fluorescence were 5.4, 5.3, 4, 2.3, and 1.3 times higher than the percentages 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 HFL1 cells that showed higher H2O2 production were 1.5, 1.6, 1.4, and I .1
Table IPercentages of exponentially growing HFLI cells that received one or more
particles% nuclear or whole cell hits by a ina of cells % of cells % change

with the 8.4- and l9-cGy irradiated medium samples, respectively (Fig. 5). Similar results were obtained with the recipient cells at the 30-mm time point (Fig. 5). Consistent with the previous observation that ROS production by cells does not require direct cell traversals by the a particles, such findings indicate that ROS-inducing activity is present in cell-free culture medium shortly after exposure to a parti des. Our findings, moreover, further suggest that ROS-inducing ac
tivity can arise from interactions between a particles and one or more

medium constituents, as well as from one or more constituents in cell-conditioned medium. Our previous study (18) revealed that exposing unirradiated HFL1
cells to a-irradiated fresh or conditioned medium pretreated with SOD

hydrogen(cGy)or dosereceiving one production0.42.5 more nuclear hits 13.41.59.2

493.620.8

receiving one or more whole-cell hits 9.2

30.4

intracellular peroxide

838.441.9 971970.8

58.2 86.9 98.9

reduced the occurrence of SCE to control levels. We therefore hy pothesized that intracellular O2@ production in unirradiated HFL1 exposed to a-irradiated (8.4 cGy) fresh and conditioned medium may also be inhibited by the addition of SOD. As summarized in Fig. 6, 100 units/ml SOD reduced the O2@ production in unirradiated HFLI
cells exposed 3966 to both a-irradiated fresh and conditioned medium by

93

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a PARTICLES

AND ROS

A
C

B
* p < 0.05

15

C C

12

Fig. 4. Intracellular H2O2 production in HFLI cells exposed

to sham-irradiated (0 cGy) and a particle-irradiated fresh me

dium (8.4 and 19 cOy) at 5 (a) incubation with the medium. significantly greater than with harvested from sham-irradiated and 30 mm (b) after the onset of Columns, means; bars, SE. *, HFLI exposed to fresh medium HFLI.

IC

6 C C

3
C C .0

8.4
Alpha dose (cGy)

19

Alpha dose (cGy)

25 and 45%, respectively. These findings suggest that O2@ may he involved in the direct or indirect mediation of excessive SCE in response to a-irradiated medium. Role of Cell.derived Factor(s) in ROS-inducing Activity after Exposure to a Particles. In addition to the role of an immediate factor or factors in inducing SCEs in unirradiated HFLI, we previ ously found that a cell-derived SCE-inducing factor or factors were present in medium harvested from HFL1 cells 24 h after a-irradiation (18). In other analyses, the activity of the cell-derived factor(s) per sisted even after freeze-thaw treatments and the medium was heated to

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

centrifuge tubes. Harvested cells were then centrifuged at 200 g for 5

mm,and the cell pelletswereresuspended in 500 pAof medium

containing DCFH-DA. The tubes were then incubated at 37C for 5

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

cytometer. Significant, dose-dependent

increases in mean DCF fluo

56Cfor 30 mm; only heating the medium to the boiling point of

water (for 1 mm) destroyed the SCE-inducing activity (18). To de termine whether a cell-derived factor or factors may cause a similar increase in intracellular ROS production, exponentially growing
HFL1 were a-irradiated in Mylar dishes at 0, 3.6, 8.4, and 19 cOy

rescence 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

after removal of culture medium. Medium was replaced on the di rectly irradiated cells, and the Mylar dishes put back into the incubator for 24 h (donors). Thereafter, the medium was then transferred onto umrradiated exponentially growing HLF1 (recipients). Recipients

interesting to note that directly irradiated cells (donors) were still capable of producing significant amounts of H,O2 in a dose-depen dent manner at 24 h (Fig. 7). Cell-free Assay. A cell-free assay was performed to determine the potential of a particles to oxidize HE to EB and DCFH to DCF,
respectively, and to determine the equivalent amounts of O2 and

* p < 0.05 @ 20 C _____

* * *

@ @

18 16 @14

.5 mm 30 mmn _____

-L-@

*1
;@Fig. 5. Intracellular H2O2 production in HFLI cells at 5 and 30 mm after

@ 12 C

c-

incubation with sham-irradiated (0 cGy) and a particle-irradiated con

ditioned medium (8.4 and 19 cGy), i.e.. medium that was exposed to a particles after removal from untreated, exponential HFLI . Columns, means; bars, SE. a, significantly higher than values obtained with HFLI

exposed to conditioned medium harvested from sham-irradiated HFLI.

.E 2
.0

8.4
Alpha Dose (cGy)

19
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a PARTICLES AND RO5

A
C 2

I
@4 15 I..

* p < 0.05
*

* *

.@ C

@
Fig. 6. Intracellular O{ production in HFL1 exposed to sham-irradiated (0 cOy) and a-irradiated (8.4 cOy) fresh (a) or conditioned medium (b) in the presence or absence of Cu,Zn
.0

sham irradiated

sham
irradiated
+

8.4 cOy

8.4 cGy
+

H20,
(200 uM)

H,O,
(200 uM)
+

SOD

SOD

(100U)

SOD (SOD)at 30 mm. Unirradiated HFLI were incubated

with sham- or a particle-irradiated medium, to which SOD was added immediately prior to transfer. Hydrogen peroxide (200
lAM) was used as a positive
5, significantly different

(100U)

SOD (100U)

control.
from HFL1

Columns,
exposed

means;
to fresh

bars,
or

SE.
con

B
C 3 C C 25

ditioned medium in the presence or absence of SOD harvested

from sham-irradiated HFLI.

I
*

*p<0.05

C
*
r

I:
H202 that may be produced in medium by the a particles. Significant, dose-dependent oxidations of HE to EB and DCFH to DCF were observed in a-irradiated MHBSS. The amounts of measured O2@ produced by 0.4-, 3.6-, and 19-cGy a particles were 15,16, and 17

r@:1
sham irradiated
+

sham irradiated

8.4cGy

8.4 cOy
+

H,O, (200uM)

II@O (2o0uk@
+

SOD

SOD (100U)

(100U)

SOD (100U)

gations have indicated that a particles can cause DNA alterations by a mechanism or mechanisms that are independent of nuclear or even whole-cell traversals (7, 8). Using the occurrence of excessive SCEs as an index of DNA damage in human lung fibroblasts, we (18) flM, respectively (Table 2). The amounts of H2O2 detected were 7, recently found that a relatively low dose of a particles results in the 8, and 9 nM at the 0.4-, 3.6-, and 19-cOy exposure doses, respectively, generation of extracellular factors, which, upon transfer to unexposed or approximately 2-fold less than a particle-induced O2@ production. normal human cells, can cause excessive SCE to an extent equivalent In comparison to these values, the concentrations of H2O2 produced in to that observed when the cells are directly irradiated with the same a similar system when HFLI were included along with the medium irradiation dose. A short-lived, SCE-inducing factor or factors are were 5060 times greater than levels produced in the cell-free generated in a-irradiated culture medium containing serum in the system (Table 3). absence of cells, and the activity of this factor or factors can be promptly inhibited by SOD. A more persistent SCE-inducing factor or factors, which can survive freeze-thawing, is heat labile and which DISCUSSION can also be inhibited by SOD, was found to be produced by fibroblasts Although direct nuclear traversals by a particles may be involved in after exposure to a particles. Such fmdings, in addition to other lines mediating their mutagenic and carcinogenic effects, previous investi of suggestive evidence, prompted us to investigate the possibility that 3968

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a PARTICLES

AND ROS

Fig. 7. Intracellular H2O2 production in recipient HFL1 cells cx posed to medium derived from HFLI exposed to 0, 8.4, and 19 cGy after 24 h. Hydrogen peroxide production was monitored in recipient HFL1 for 30 mm and 3 h. Directly irradiated (donors) HFL1 cells were still capable of producing significant amounts of H2O2 in a dose

dependent mannerat 24 h. Columns, means;bars, SE. a, significantly

different from values obtained with HFL1 incubated in medium har

vested from sham-irradiated HFL1.

DONORS

(30 MIN)

RECIPIENTS

(30 MIN)

DONORS

(3 Lw)

RECIPIENTS (3 Lw)

Table 2 Superoxide and hydrogen peroxide production measured on a

spectrofluorometer in a cell-free system

cOy, resulted in time-dependent

increases in H2O2 production over a

45-mnperiodbeforesubsiding, whereas H2O2 valuesforthehigher

doses peaked either at 15 or 30 mm postexposure before showing declines. We presently do not have a firm, mechanistic explanation for these complex H202 responses, but it is likely that they involve numerous underlying and interrelating components that can contribute to the formation and removal of H2O2, e.g. , intracellular status of SOD, iron, and catalase (3840). Regardless, our collective results suggest that the a particle-induced ROS response involves mainly the plasma membrane-bound NADPH oxidase complex, in that the re sponse was found to be inhibitable by diphenyleneiodonium, a selec tive inhibitor of the plasma membrane NADPH oxidase (41). In addition to implicating the generation of O{ as an inherent compo nent of the response, this finding, along with results obtained with the mitochondrial inhibitor sodium azide, shows that the increases in intracellular O2@ and H,O2 were not merely due to an enhanced state of autoxidation of chemically reactive components produced during reductive processes that are associated with the mitochondnal electron transport system (42). As with the induction of excessive SCE ( 18), the ROS response
does not require nuclear or whole-cell hits by the a particles for its

a production(cOy)(aM) doseO2@ 0.4 3.6 19

14.9 0.05

H2O2 production
(aM) 6.9 0.06a

15.7 o.o2a
16.80.10

8.2 0.29
8.7 0.09

a p < 0.05;significantly differentfromsham-irradiated controls(n

3).

the extranuclear effects of a particles may be related to an a particle associated production of ROS. Here, we have presented new evidence that the intracellular pro duction of O2' and H202 is indeed increased following exposure to low doses of a particles. Moreover, our analyses indicate that the induction of this ROS response does not require direct nuclear or even whole-cell hits by a particles. Furthermore, as in our previous inves
tigation (18), in which SCE-inducing activity was found in cell-free

a-irradiated medium and in supernatants from a-irradiated cells, we have shown that SOD-inhibitable, ROS-inducing activity is also pres ent in such samples. Our initial experiments involved measuring the intracellular ROS inducing activity of a particles in terms of post exposure time and dose. Even at the lowest dose studied (0.4 cOy), intracellular O2@ significantly increased in cells shortly after exposure to the a parti des. Enhancements in the rates of O2@ production above basal levels, however, were short-lived, i.e. , they ceased within 15 mm after exposure. Interestingly, the a particle-induced increases in O2@ did not consistently scale with increasing exposure dose, but, instead, the induction of O2@ seemingly occurred in an all-or-none fashion within the dose range we examined. Of possible mechanistic rele vance, a virtual constancy in the mean number of induced SCEs/cell, independent of the dose of a-particles administered, was also ob served previously, once a dose threshold was reached (8). The intracellular production of H2O2 as a function of dose and postexposure time, on the other hand, was much more complex. Some evidence for a dose-response relationship was observed as of 15 mm after exposure over the 0.43.6-cOy dose range. However, such a
relationship did not continue with the higher 8.4- and 19-cOy doses,

initiation. It can be induced by soluble, transmissible factors that are generated by the interaction of a particles with serum-containing culture medium or with cells. How the NADPH oxidase complex becomes activated in response to exposure to a particles and the transmissible factor(s) is unclear. We did observe that the a particle induced oxidative burst in the fibroblasts could be inhibited by DMSO, a potent scavenger of OH radicals (23, 43). Although it is well recognized that the presence of iron or other transitional metal cations can convert O2 and H,O, to @OH downstream of activation of NADPH oxidase (44), the possibility that OH may initiate activa tion of NADPH oxidase, perhaps as a consequence of lipid peroxida tion, cannot be ruled out (45). Nor can we rule out the possibility that the NADPH-oxidase complex is activated by O2@ or H2O2. Hydroxyl radicals generated as a radiolytic product during the exposure of
Table 3 Hydrogen peroxide production measured on a spee:rofluorome:erfrom (I X 1O@ cells) exposedto a particles (nM)0.4385 a dose (cOy)H,02 production HFLJ

which actually resulted in lesser amounts of H2O2 at the 15 mm postexposure time point than what was observed with the lower 3.6-cOy dose. In terms of the postexposure kinetic patterns of H2O2 production, exposure to the lower doses of a particles, i.e. , 0.4 and 1.5 3969

ID3.6430 719562
IIa

control (a = 3). p < 0.05;significantly different sham-irradiated

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a PARTICLES ANDROS medium to a particles would be expected to be short-lived, with a z@ of l0@ 5 (46); no transmissible ROS-inducing activity from this source of @OH would be expected. It may be possible, however, for 0H to be produced in the medium in a more sustained manner. For example, one mechanism for a more persistent production of OH could begin with the generation of O2@, which can arise from the interaction of molecular oxygen with either electrons that are ejected from water by ionizing radiation or with the radiolytic product if
@ (Caq + 02 O2@; if + 02 @ O2@ + H@; Ref. 47). Conversion to

@OH could then proceed via an iron-catalyzed Haber-Weiss reaction, i.e., O2@ + H2O2 i 02 + OH + OH (48), after dismutation of some O2@ to H2O2, which, expectedly, would be facilitated by extracellular SOD that was present in serum (4951). Hydroxyl radicals from this source, in addition to 0H initially produced radio lytically, in turn could form a second source for the further production of O2@ and, subsequently, the further production of OH. According

to this scheme,@OH radicalsabstracthydrogenatomsfrom thecarbon

chains of unsaturated fatty acids present in serum or cell membranes to yield peroxy radicals. Further hydrogen abstraction by the peroxy radicals results in the formation of lipid peroxides, which also can initiate a chain reaction leading to peroxidation of a large number of unsaturated fatty acids. It is well established that lipid peroxidation can simultaneously lead to the generation of numerous metabolites that have the propensity to induce O2@ generation (5254).Further @OH generation would then follow, as described before, with repetition

ROS in cells from individuals with the cancer-prone disorder Bloom's syndrome (55, 63). Conceivably, the induction of SCE by a particles may at least in part reflect an alteration a cell's state of oxidant antioxidant imbalance. With regard to carcinogenesis, substantial cv idence exists in support of ROS as mediators of radiation-induced damage in a variety of systems (64). ROS cause formation of oxidized bases and a spectrum of DNA lesions including base damage, single strand breaks, double-strand breaks, cross-linking of DNA, and dam age to the deoxyribose moiety (6470). Other lines of evidence indicate that ROS play important roles in virtually all stages of tumorigenesis initiated by ionizing radiation (65). McLennan et a!. (70) demonstrated some time ago that OH radicals generated from 02 H2O2, and iron were the agents responsible for oxygen-induced cellular damage and death (70), and more recent studies have shown that exposure of eukaryotic cells to radiation is associated with in duction of oxidative stress and attendant DNA damage, inter a!ia (48). Extension of our present findings to the in vivo condition would suggest that inhaled radon and radon progeny may induce a condition of oxidative stress that is transmissible among lung cells and that may be involved in mediating DNA damage. ACKNOWLEDGMENTS We express our gratitude to A. Deshpande, Y. Valdez, and Y. Shou for their technical assistance during the course of this study. REFERENCES
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