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Original Contribution

Induction of bystander effects by UVA, UVB, and UVC radiation

in human broblasts and the implication of reactive oxygen species

Maria Widel n, Aleksandra Krzywon, Karolina Gajda, Magdalena Skonieczna,Joanna Rzeszowska-Wolny

Biosystems Group, Institute of Automatic Control, Faculty of Automatics, Electronics, and Informatics, Silesian University of Technology,

44-100 Gliwice, Poland

a r t i c l e i n f o

Article history:

Received 21 June 2013

Received in revised form

5 December 2013

Accepted 18 December 2013Available online 27 December 2013

Keywords:

Ultraviolet radiation

Bystander effect

Humanbroblasts

Apoptosis

Premature senescence

Reactive oxygen and nitrogen species

Mitochondrial membrane potential

Interleukins 6 and 8

Free radicals

a b s t r a c t

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells

induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well

studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far

have been reported mainly after UVA and UVB radiation. The studies described here were aimed at

comparing the responses of human dermal broblasts exposed directly to UV (A, B, or C wavelength

range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation

system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of

senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide

radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into

the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of

ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival

and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects

differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in

generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in

neighbors. The level of cellular ROS increased signicantly shortly after UVA and UVB exposure,

suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation.Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells

and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly

associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but

not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander

effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments.

The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also

in neighboring cells, and generation of proinammatory cytokines, processes entailing cell damage

(decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential

hazard for human health, not only due to direct mechanisms, but also due to bystander effects.

& 2013 Elsevier Inc. All rights reserved.

Radiation-induced bystander effects, which appear in nontar-geted cells mainly as cell-damaging events (decreased viability,

reduction of clonogenic survival, induction of apoptosis, and

cytogenetic damage), are well known phenomena in the case of

ionizing radiation[14], but knowledge of bystander effects after

ultraviolet radiation (UVR) is limited. UVR comprises three differ-

ent wavelength bands, long-wave UVA (320400 nm), middle-

wave UVB (290320 nm), and short-wave UVC (200290 nm)

[5,6]. The main source of UVR in the environment is solar radiation,

of which about 95% is UVA and 5% is UVB; UVC is almost completelyabsorbed in the upper part of the stratosphere [5]unless it traverses

an ozone hole in this layer. Bystander effects [79] and related

genomic instability [10,11] have been reported after UVA and UVB

radiation, but very limited data are available on UVC-induced bystan-

der effects, probably because of less interest because this wavelength

does not reach the earth. The short-wave radiations (UVB under

300 nm and UVC) are especially dangerous for cells because their

bands coincide with the absorption spectra of DNA, RNA, and proteins

and they can damage DNA by forming cyclobutane pyrimidine dimers

(CPDs) and 6-4 photoproducts (6-4 PPs), which can lead directly or

indirectly to DNA strand breaks[1214]and possibly to mutation and

neoplastic transformation[15].

Contents lists available atScienceDirect

journal homepage: www.elsevier.com/locate/freeradbiomed

Free Radical Biology and Medicine

0891-5849/$- see front matter& 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.021

n Corresponding author. Fax:48 32 237 2127.

E-mail address: [email protected](M. Widel).

Free Radical Biology and Medicine 68 (2014) 278287
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UVR is responsible for the induction and promotion of basal

and squamous cell skin cancer [16] and is also an important

etiological factor in malignant melanoma [17,18]. Data about the

possible signicance of bystander effects in UV carcinogenesis

come exclusively from in vitro studies. Human keratinocytes

whose precursor generations were exposed to UVA showed a

reduction of clonogenic cell survival [9] and persistent genomic

instability [7]. A reduction of clonogenic cell survival, genomic

instability, and delayed mutation has been also observed inbystander Chinese hamster broblasts after exposure to UVA and

UVB [10,11]. Furthermore, apoptosis was observed in bystander

human keratinocytes after both UVA and UVB exposure [8],

although in another study [19] no bystander effect was found

after UVB radiation.

Bystander effects induced by ionizing radiation [2022]as well

as ultraviolet radiation [7,11,23] are reduced in the presence of

antioxidants and may be linked to oxidative stress. Each region of

the ultraviolet spectrum induced the formation of 8-oxo-7,8-

dihydro-20-deoxyguanosine in calf thymus DNA and in HeLa cells

in a uence-dependent manner, with singlet oxygen (1O2) playing

the predominant role [24]. UVC induced DNA double-strand

breaks measured by-H2AX and 53BP1 foci formation in bystan-der humanbroblasts more effectively than in irradiated cells, and

this effect was mediated by reactive oxygen species (ROS) [23].

Here we report that all three UV wavelength bands, UVA, UVB, and

UVC, induce bystander effects in human dermal broblasts with a

pattern of responses that differs for each. Studies of the levels of

ROS and reactive nitrogen species (RNS) and changes in mitochon-

drial membrane potential suggest that ROS are implicated in this

induction. It is known that ROS induced by UV radiation can

damage DNA and lead to various skin diseases and carcinogenesis

[reviewed 25]. The high production of interleukin 6 (IL-6), a

mediator of inammation, points to its possible role in the

induction of UV radiation-induced bystander effects. Although

further studies are required to gain knowledge of the detailed

nature of the mediators and their interactions, the present results

suggest that all bands of UVR carry a potential hazard for human

health not only due to direct mechanisms, but also due to

bystander effects.

Materials and methods

Cells and experimental procedure

Neonatal human dermal broblasts (NHDF-Neo, Lonza, Poland)

in early (1013) passages were grown in Dulbeccos modied

Eagles medium/Nutrient Mixture F-12 Ham medium (Sigma

Aldrich), supplemented with 12% fetal bovine serum (PAA, Immu-

niq, Poland) and 80 mg/ml gentamycin (Krka, Poland). Irradiated

and nonirradiated cells were co-incubated in six-well dishes with

an insert separating the two cell populations by a 0.4-mm-poremembrane (BD Immunogen) to allow diffusion of medium com-

ponents between them, as described previously [26]. About 20 h

before irradiation cells were seeded into wells (1 105 cells/well

in 2 ml medium) and those not to be irradiated (bystander cells)

were seeded on inserts. Before irradiation the medium was

removed and the cells in wells were irradiated (covers opened)

at room temperature (21 1C) with various doses of UVA (365 nm),

UVB (302 nm), or UVC (254 nm) generated by UV crosslinkers (CL-

1000 models, UVP, Upland, CA, USA). We used doses of 520 kJ/m2

(UVA), 210 kJ/m2 (UVB), and 10200 J/m2 (UVC). Immediately

after irradiation, 2 ml of fresh medium was added to the wells, and

then the inserts, also with medium changed, were inserted and

the cells were cocultured in a CO2incubator (standard conditions:

5% CO2, 80% humidity, 37 1C) for the desired period.

Cell survival assays

The proportion of viable cells was determined using the 3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-

phenyl)-2H-tetrazolium salt (MTS) reduction assay (Cell Titer 96

AQueous One Solution Cell Proliferation Assay, Promega). MTS is

bioreduced by the dehydrogenase enzymes present in live, meta-

bolically active cells to the colored product formazan. The quantity

of formazan measured by the absorbance is directly proportionalto the number of surviving cells. The manufacturer0s protocol was

adapted for our experimental system. Briey, cells were harvested

separately from wells and inserts by trypsinization, spun down,

washed in phosphate-buffered saline (PBS), and loaded with MTS

reagent. The suspensions were transferred to 96-well plates and

incubated for 60 min in a humidied CO2 incubator, and absor-

bance was measured at 490 nm using a universal plate reader

(Epoch, Biotek Instruments). Survival of control, irradiated, and

bystander cells is presented as mean absorbance7SD from three

independent experiments.

Apoptosis and necrosis assays

Apoptosis and necrosis were assessed by ow cytometry using

the Dead Cell Apoptosis Kit with annexin VFITC and propidium

iodide (PI; Invitrogen). Annexin V is bound to phosphatidylserine,

which is translocated from the inner to the external membrane

layer at an early stage of apoptosis [27]. Cells were exposed to

20 kJ/m2 UVA, 10 kJ/m2 UVB, or 200 J/m2 UVC and co-incubated

with unexposed cells for the desired time. Cells were harvested

separately from wells and inserts, spun down, washed with PBS,

suspended in staining buffer, and incubated for 15 min with

annexin VFITC according to the manufacturer0s protocol. PI,

which stains necrotic cells, was added and the distribution of

living, apoptotic, and necrotic cells was measured by ow cyto-

metry (BD FACSAria III) using excitation/emission maxima of

494/518 nm for annexin VFITC and 535/617 nm for PI. Ten thou-

sand cells were counted. Results are presented as mean uores-

cence intensities7SD from three independent experiments.

ROS assay

Total cellular ROS were assayed as described elsewhere [26]

using 20,70-dichlorouorescein diacetate (DCFH-DA; Sigma), which

was deacetylated to nonuorescent DCFH by intracellular

esterases and then converted by cellular ROS to oxidized, uor-

escent DCF. After co-incubation for 3, 6, 12, or 24 h, irradiated and

bystander cells were harvested, suspended in growth medium,

and loaded with DCFH-DA (nal concentration 30 mM) for 30 min

at 37 1C in the dark. After the cells were washed in PBS to remove

extracellular dye, suspended in PBS, and incubated for 15 min on

ice in the dark, ROS were determined in 10,000 cells by ow

cytometry using the FITC conguration with excitation and emis-sion wavelengths of 488 and 530 nm, respectively. Results are

expressed as mean uorescence intensities7SD from three inde-

pendent experiments.

Measurement of superoxide radical anions

The MitoSOX red mitochondrial superoxide indicator (Invitro-

gen), which permeates into live cells and selectively targets

mitochondria[28,29], where it is rapidly oxidized by superoxide

but not by other ROS or RNS and emits red uorescence, was used

according to the manufacturer0s protocol. Cells from wells and

inserts were harvested separately, spun down, suspended in

medium, loaded with MitoSOX (nal concentration 5 M), and

incubated for 20 min at 37 1C. Fluorescence was measured in

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10,000 cells by ow cytometry using the phycoerythrin cong-

uration (488-nm laser line, LP mirror 566, BP lter 585/42). Results

are presented as mean uorescence intensities7SD from three

independent experiments.

Measurement of mitochondrial membrane potential

Cells harvested by trypsinization were washed in PBS, suspended

in fresh growth medium, and loaded with 50 nM tetramethylrhoda-mine ethyl ester (SigmaAldrich), a mitochondrial membrane-specic

agent[30]. After 30 min incubation at 37 1C they were washed in PBS

to remove extracellular dye, resuspended in PBS, and analyzed by ow

cytometry with excitation and emission at 547 and 585 nm, respec-

tively; 10,000 cells were counted. Results from three independent

experiments are presented as means7SD (in arbitrary units related to

uorescence intensity).

Assay of nitric oxide

The indicator 4-amino-5-methylamino-20,70-diuorescein dia-

cetate (DAF-FM diacetate; Invitrogen), which is deacetylated to

DAF-FM by intracellular esterases and emits uorescence at

excitation/emission maxima of 495/515 nm when it reacts withnitric oxide (NO), was used to measure intracellular NO[31]. Cells

harvested from wells and inserts were suspended in growth

medium and incubated with DAF-FM (nal concentration 1 M)for 30 min at 37 1C and the uorescence intensity was measured in

10,000 cells by ow cytometry with the same conguration as for

ROS assays. Results are presented as mean uorescence intensi-

ties7SD from three independent experiments.

Determination of superoxide dismutase (SOD) activity in cell extracts

SOD activity was determined by a colorimetric method using a

High Throughput Superoxide Dismutase Assay Kit (Trevigen,

supplied by Biokom, Poland). Briey, cells were harvested sepa-

rately from wells and inserts by trypsinization, washed with coldPBS, and centrifuged at 250gfor 10 min at 4 1C. The pellets of cells

were lysed on ice for 30 min in cell extraction buffer contained in

the kit and centrifuged at 10,000gfor 10 min at 4 1C. Supernatants

were transferred to tubes precooled at 80 1C and frozen at

80 1C until SOD measurement. Measurement was performed

according to the protocol provided by the manufacturer. SOD

activity per microgram of protein was calculated and data are

presented as percentage change in relation to the control taken as

100% at a given point in time; the values for the control samples

were in the range from 0.04 to 0.065 U/g protein.

Determination of IL-6 and IL-8 in culture media

Two series of experiments were done, one with cells irradiated and

incubated without cells in the inserts, whereas in the second cong-

uration irradiated cells were co-incubated with bystander cells in

inserts. The media were collected (in bystander conguration media

from wells and inserts were collected together), centrifuged (250g,

10 min, 4 1C) to remove any cells and particles, and immediately

stored at 201C until the cytokine assay. IL-6 and IL-8 concentrations

were determined by the quantitative sandwich enzyme-linked immu-

nosorbent assay (ELISA), using immunoassay kits (R&D Systems,

supplied by Biokom). The ELISAs were performed according to the

manufacturer0s protocol using 96-well plates coated with antibodies to

IL-6 or IL-8. Optical density of the standard solutions and the samples

was measured at 450 nm using a microplate reader. Standard curves

were generated and concentrations of interleukins in samples were

calculated and expressed in pg/ml.

Analysis of senescent cells

Analysis of senescent cells [32] was based on -galactosidaseexpression using a Senescence Cells Histochemical Staining Kit

(SigmaAldrich). Cells growing in wells (irradiated) and in inserts

(bystander) were stained in situ after 24 h co-incubation according to

the manufacturer0s protocol. Estimation of senescence frequency was

performed using an inverted microscope (Zeiss, Germany) and count-

ing at least 1000 cells. Three independent experimental sets (well-

sinserts) were assayed and data are presented as means7SD.

Results

Survival of irradiated and bystander cells

Normal human dermal broblasts were used in these experiments

because they represent one of the cell types present in dermal tissue,

which is regularly exposed to UV radiation. To obtain comparable

results for three bands of UV radiation that have different energies,

we chose different dose ranges of 520 kJ/m2 for UVA, 210 kJ/m2 forUVB, and 50200 J/m2 for UVC, partly based on doses used in

published studies concerning genomic instability [10,11] and DNA

damage[33,34]. The wavelength range of UVA used was lower than

that used in other studies of bystander effects and genomic instability

[e.g., 79]. The survival of control, UV-exposed, and bystander

broblasts is presented in Figs. 13. The survival of control cells

growing in wells and in inserts was similar, and therefore common

control curves are presented. The measurement of survival was started

from 15 min to allow for at least a short co-incubation of irradiated

with bystander cells.

15 min 24 h 48 h 72 h

20 kJ/m2

15 min 24 h 48 h 72 h

Incubation time

10 kJ/m2

CT IR BY

0

0.8

1

15 min 24 h 48 h 72 h

Absorbance

UVA 5 kJ/m2

0.6

0.4

0.2

Fig. 1. Survival of NHDF broblasts estimated by MTS assay; CT, control cells; IR, cells exposed to various doses of UVA; and BY, unexposed bystander cells co-incubated with

them. Results are expressed as mean absorbance7SD from three independent experiments.

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UVR-exposed cells showed a decline in survival with time and with

increasing dose, which was seen clearly after 72 h for all three

wavelengths, whereas survival of control cells increased with time

because of proliferation. The highest survival at 72 h was observed

after exposure to UVA (50% after 20 kJ/m2,Fig. 1), whereas for UVB

(10 kJ/m2) viability dropped to10% (Fig. 2) and for UVC(200 J/m2) to

20% (Fig. 3). Bystanders of UVA-exposed cells show a diminution of

survival with increasing time compared to controls, which at 48 h was

even more marked than for irradiated cells (Fig. 1).

In the cases of UVB (Fig. 2) and UVC (Fig. 3), the survival of

bystander cells measured at 72 h dropped after the highest doses

to about 60 and 50% of control, respectively.

Apoptosis and necrosis

To obtain reliable quantitation of apoptotic cells and of ROS and

RNS in cells, the highest UVR doses and the time span of 24 h were

used. The frequencies of apoptosis in control cells growing in wells and

in inserts were comparable (4%), and a common control is shown

(Fig. 4). UVA (20 kJ/m2) caused a statistically signicant approximately

twofold increase in the frequency of apoptotic cells, which persisted at

a slightly lower level up to 12 h. Apoptosis also appeared in UVA

bystander cells but with some delay, reaching a greater than twofold

increase by 6 h (Fig. 4a). UVB (10 kJ/m2) induced a signicant greater

than twofold increase in apoptosis by 12 h and an approximately

vefold increase by 24 h (Fig. 4b). In UVB bystander cells the apoptosis

frequency increased slightly, although signicantly, after 3 h and

persisted at a comparable level up to 24 h (Fig. 4b). UVC (200 J/m2)

induced apoptosis in irradiated cells with a frequency nearly twice

than in control cells at 3 h, which slightly decreased at 6 h and

dropped almost to the control level after 12 h. However, in UVC-

irradiated cells apoptosis increased again after 24 h, probably as a

delayed consequence of the ROS elevation seen at 12 h (Fig. 4c). In

bystander cells the apoptosis frequency did not change signicantly

except for a small but signicant increase at 6 h (Fig. 4c).

The insets in Fig. 4 show the cell survival measured by MTS

assay at the same time points at which apoptosis was assessed.

For UVA, the data are quite consistent with the data for apoptosis

at 6 and 12 h; the increase in apoptosis, particularly in bystander

cells, is accompanied by a considerable reduction in survival

(Fig. 4a). The good agreement between apoptosis and survival

can also be seen at 12 and 24 h for UVB and UVC (Figs. 4b and c).

Neither UVA nor UVB induced necrosis within the experimental

period, but interestingly UVC (200 J/m2) induced a low frequency(0.5%, not signicantly different from the control) after 24 h (data

not shown).

Cell senescence

Recently, attention has been paid to stress-induced premature

senescence [3538]. Hallmarks of senescent cells include an essentially

irreversible growth arrest and expression of senescence-associated

-galactosidase and p16INK4a. We evaluated the expression of-galactosidase, the known marker of senescence [32], after stainingthe cells in situ in wells and inserts after 24 h co-incubation.

The microscopic analyses indicated a large difference in the

efciency of senescence induction by various UV bands. It was striking

that UVC induced senescence very effectively in both irradiated and

15 min 24 h 48 h 72 h15 min 24 h 48 h 72 h

Incubation time

CT IR BY

15 min 24 h 48 h 72 h

Ab

sorbance

10 kJ/m25 kJ/m

2

0

0.8

1

UVB 2 kJ/m2

0.6

0.4

0.2

Fig. 2. Survival of NHDF broblasts estimated by MTS assay; CT, control cells; IR, cells exposed to various doses of UVB; and BY, unexposed bystander cells co-incubated with


15 min 24 h 48 h 72 h

Absorbance

15 min 24 h 48 h 72 h

Incubation time

CT IR BY

15 min 24 h 48 h 72 h

200 J/m2100 J/m

2

0

0.8

1UVC 50 J/m

2

0.6

0.4

0.2

Fig. 3. Survival of NHDF broblasts estimated by MTS assay; CT, control cells; IR, cells exposed to various doses of UVC; and BY, unexposed bystander cells co-incubated with



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neighboring cells (Fig. 5). In experiments using UVB, senescence was

induced in bystander cells, whereas in directly exposed cells an

extremely high yield of apoptosis was seen at that time (Fig. 4b). In

contrast, UVA generated senescence in directly exposed cells, but not

in bystanders. These differences suggest that the signals secreted bycells in response to different UV bands differ, although their nature

and interactions remain to be elucidated.

ROS levels

In cells irradiated with UVA (20 kJ/m2) the level of total cellular

ROS increased to almost threefold that in control cells by 3 h

(Fig. 6a), and in bystander cells the increase was approximately

twofold by 3 and 6 h. After 12 h the ROS level in both irradiated

and bystander cells decreased to the control level, followed by a

second but statistically insignicant increase after 24 h (Fig. 6a).

UVB (10 kJ/m2) was somewhat less effective than UVA (20 kJ/m2);

the yield of ROS reached almost twofold the level in control cells at

3 h and was even higher in bystander cells (Fig. 6b). It is

interesting that UVC increased the level in bystander cells sig-

nicantly, especially after 12 h, although it did not generate such a

highly signicant amount of ROS in irradiated cells (Fig. 6c). The

ROS level in control cells also increased after 24 h. We suppose

that undistorted proliferation of cells in the wells and the inserts

leads to faster acidication of medium and changes in the micro-

environment, which result in increased ROS generation.

Superoxide radicals

UVA (20 kJ/m2) generated a signicant level of superoxide at

3 and 6 h in irradiated cells (Fig. 7a), whereas after UVB exposure(10 kJ/m2) a signicant increase was observed after 24 h (Fig. 7b).

The highest level of superoxide was generated by UVC (200 J/m 2);

it reached 5-fold the control level after 3 h and then rapidly

declined (Fig. 7c), and a relatively high level of superoxide (2.5-

fold the control) was measured in bystander cells at 3 h.

Mitochondrial membrane potential

UVA (20 kJ/m2) led to a signicant decrease in mitochondrial

membrane potential in irradiated cells between 3 and 12 h

(Fig. 8a). In bystander cells the potential increased by 12 h and

then decreased to under the control level by 24 h. At this time

point the mitochondrial potential in UVA-exposed cells increased,

but was still signicantly lower than in control (Fig. 8a).After UVB (10 kJ/m2) the potential showed a rather stable level in

exposed cells and someuctuation in bystanders, e.g., a slight increase

at 3 h and a decline to below the control level at 6 h (Fig. 8b). In

UVC-exposed cells the membrane potential had increased at 12 h but

was comparable to that in control cells at 24 h, whereas in bystanders

the mitochondrial membrane potential remained constant (Fig. 8c).

Nitric oxide

The level of cellular NO did not change markedly after exposure to

any of the wavelength bands of UVR during the whole range of time,

with the exception of 12 h, at which it was reduced in bystander cells

after UVA and UVB, and a signicant reduction in UVC-exposed cells

and their bystanders was seen (Fig. 9).

0

5

10

15

20

25

3 6 12 24

Apoptoticcells(%

total)

3 6 12 24

Incubation time (h)

UVB

Ct IR BY

3 6 12 24

UVA UVC

Fig. 4. Frequency of apoptotic cells in control cultures (Ct), cells exposed to UV (IR), and unexposed bystander cells co-incubated with cells exposed to UV (BY). Doses were

UVA, 20 kJ/m2; UVB, 10 kJ/m2; and UVC, 200 J/m2. Data are means7SD from three independent experiments. nSignicant difference from the control level of apoptotic cells

(po0.05, Studentsttest). The insets show survival assay data obtained from MTS assays at the same time points and doses used for apoptosis assays.

0

2

4

6

8

10

Percentageofsenescentcells

Fig. 5. Percentage of senescent cells in wells and inserts after 24 h co-incubation

assessed on the basis of expression of-galactosidase. Data are means7SD from

three independent experimental sets (wellsinserts) in which 1000 cells were

counted. nStatistically signicant difference from the control at po0.05 (Studentst

test). UV doses were the same as those in Fig. 4.


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SOD activity

To check whether the generation of reactive oxygen species,

including superoxide, entails changes in the activity of one of the

key antioxidant enzymes, superoxide dismutase, SOD activity

measurements were done in cell extracts soon after irradiation

and after 3, 12, and 24 h of exposure. Whereas SOD activity in

control cells was at a fairly even level (0.04

0.06 U/g protein),

3 6 12 24

Incubation time (h)

Ct IR BY

3 6 12 24

0

500

1000

1500

2000

2500

3 6 12 24

ROSlevel(a.u.)

UVBUVA UVC

Fig. 6. Intracellular ROS levels in control cells (Ct), cells exposed to UVR (IR), and unexposed bystander cells co-incubated with cells exposed to UVR (BY). Data are means7SD in

arbitrary units (a.u.) from three independent experiments. nSignicant difference from the control level (po0.05, Studentsttest). UV doses were the same as those shown in Fig. 4.

0

500

1000

1500

2000

3 6 12 24

Superoxidelevel(a.u

)

3 6 12 24

Incubation time (h)

Ct IR BY

3 6 12 24

UVBUVA UVC

Fig. 7. Superoxide radicals in control cells (Ct), cells exposed to UVR (IR), and unexposed bystander cells co-incubated with cells exposed to UVR (BY). Data are means7SD in

arbitrary units (a.u.) from three independent experiments. nSignicant difference from the control level of superoxide radicals (po0.05, Studentsttest). UV doses were the

same as those shown in Fig. 4.

0

500

1000

1500

3 6 12 24

mit

(fluorescencea.u.)

3 6 12 243 6 12 24

Incubation time (h)

Ct IR BY

UVBUVA UVC

Fig. 8. Mitochondrial membrane potential expressed as uorescence signal in arbitrary units in control cells (Ct), cells exposed to UVR (IR), and unexposed bystander cells

co-incubated with cells exposed to UVR (BY). Data are means7SD from three independent experiments. nSignicant difference from the control level (po0.05, Studentst

test). UV doses were the same as those in Fig. 4.


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exposure of cells to all UV bands caused upregulation of this

enzyme practically immediately after irradiation, but to varying

degrees (Fig. 10).

UVA at a dose of 20 kJ/m2 was most effective, and after 15 min

a twofold increase in activity was observed, which after 3 h

reached a level more than ve times higher than the control

(Fig. 10a). At this time SOD activity was also doubled in bystander

cells, but later the activity of SOD declined even to under the

control level in exposed and bystander cells. UVB also caused

upregulation of SOD very quickly in irradiated and bystander cells,

which peaked at 3 h (Fig. 10b). In UVC-exposed cells a slightincrease in SOD activity was seen between 15 min and 12 h, and a

signicant increase to about threefold of the control by 24 h. The

increase in SOD activity in bystander cells was also shifted in time

to 12 and 24 h (Fig. 10c).

Generation of interleukins

Measurement of IL-6 and IL-8 cytokines as potential mediators

of bystander effect was performed in culture medium collected

from irradiated cells incubated alone or co-incubated with bystan-

der cells. InFig. 11we see that the concentration of IL-6 increased

immediately after irradiation with all UV wavelengths under both

experimental conditions and remained signicantly higher com-

pared to the control until 6 h. UVA seemed to be most effective,

but although UVA induced IL-6 to comparable levels in both

systems, the level of IL-6 in UVB and UVC co-incubation systems

increased signicantly, which indicates that IL-6 must be also

generated by bystander cells. A signicant decrease in the level of

IL-6 by 24 h was associated with an increase in apoptosis at

this time.

In the case of IL-8, production of this cytokine over the control

level was observed only for UVA in an experimental system

without co-incubation (Fig. 11c). In UVB and UVC the IL-8

concentration in culture medium was permanently below the

control level. However, in the co-incubation system the IL-8concentration in the medium was lower than in the control for

all three bands of UV and this reduction was statistically signi-

cant between 15 min and 6 h.

Discussion

In this study we investigated the occurrence of bystander

effects in normal human broblasts after exposure to three

different UV bands and we looked for differences and similarities

in the effectiveness at reducing the viability of cells, inducing

apoptosis and senescence, and generation of putative mediators.

Normal human broblasts exposed to any of the three bands of

ultraviolet radiation, UVA (365 nm), UVB (302 nm), and UVC

0

300

600

900

3 6 12 24

Nitrico

xide(a.u.)

3 6 12 24

Incubation time (h)

CT IR BY

3 6 12 24

UVBUVA UVC

Fig. 9. Nitric oxide levels in control cells (CT), cells exposed to UVR (IR), and unexposed bystander cells co-incubated with cells exposed to UVR (BY). Data are means7SD in arbitrary

units (a.u.) from three independent experiments. nSignicant difference from the level in control cells (po0.05, Studentsttest). UV doses were the same as those in Fig. 4.

0

100

200

300

400

500

600

700

800

15 min 3h 12 h 24 h

RelativeSODactivity

15 min 3 h 12 h 24 h

Incubation time

Ct IR BY

15 min 3 h 12 h 24 h

UVBUVA UVC

Fig.10. The relative activity of superoxide dismutase in cell extracts, expressed as a percentage of controls at each time point (the measured values for control samples werein the range from 0.04 to 0.065 U/g protein). Data are the means7SD from measurement of two independent samples performed in duplicate for each time point.nStatistical signicance at po0.05 (Studentsttest). UV doses were the same as those in Fig. 4.


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(254 nm), induced bystander effects manifested as a reduction in

cell survival and an increased frequency of apoptosis in cells of the

same type separated from them by a 0.4-mm-pore membrane,

which allowed diffusion of medium components. UVA appeared to

be relatively the most effective, even at doses well below the

minimal erythema dose (MED; 1 MED corresponds to 750 kJ/m2

[39]). The highest dose of UVA we used, 20 kJ/m2, reducing cell

survival and inducing apoptosis in directly exposed and bystander

broblasts, was low compared to that (100 kJ/m2) which reduced

clonogenic cell survival in bystander human keratinocytes and

broblasts [19]. UVB at 10 kJ/m2, reducing survival even more

efciently, was equivalent to 1 MED [39]. This dose was higher

than that used in a similar co-incubation system [19] in which

400 J/m2 did not induce a bystander effect, but it corresponds to

those used in other studies of bystander effect [11]and chromo-

somal damage[34]. Published data on bystander effect induction

after UVB exposure are not consistent and may depend on the cell

type, the evaluation system, the dose, and the endpoints; for

example, an increased apoptosis frequency was reported in

bystander keratinocytes exposed to 300 J/m2 UVB[8], a dose lower

than that used in[19], which did not induce a bystander effect. In

our study, UVB at 10 kJ/m2 was extremely effective at inducingapoptosis in irradiated cells and showed a similar effect in

bystander cells (Fig. 4b). UVC at 200 J/m2 reduced the survival of

bystander cells even more effectively than UVB at 10 kJ/m2

(compare Figs. 2 and 3) and was effective in apoptosis induction

in irradiated cells, but not in bystander cells (Fig. 4c). At the same

time UVC radiation induced efciently senescence in the irradiated

and neighboring cells (Fig. 5). This increase in senescence appears

to be associated with increased levels of ROS, but secretion of IL-6

by senescent cells seems probable.

The mechanism of action of UVR on cells is different for the

three wavelength bands [14;reviewed in 40]. UVA acts mainly

through generation of ROS such as singlet oxygen and hydroxyl

radicals, which can induce oxidative damage to DNA, proteins, and

lipids [7,9,14]; UVB also generates ROS [8,11], but UVC rather

damages DNA directly by forming CPDs and 6-4 PPs [14]. Our

observations of ROS generation in cells exposed to UVA and UVB,

but less effectively by UVC, are consistent with this picture.

Different types of cellular damage induced by different UV bands

may start the cellular response from different signaling pathways,

and because of that one could expect responses varying in

efciency and kinetics. However, many characteristics of cellular

response to different UV bands are very similar. For all three bands

of UV wavelengths bystander effects appear after relatively low

doses, all induce signaling to nonirradiated neighbors and a

decrease in their survival, all induce SOD activity that correlates

with the increase in ROS.

Several cytokines may be implicated in the bystander effect,

including transforming growth factor and tumor necrosis factor [23]. The proinammatory cytokines IL-6 and IL-8 have been also

proposed as signaling molecules in bystander effects because they

were detected in the medium after ionizing radiation[41,42]and UVB

radiation[43]. In our experiments we observed an increase in IL-6 in

the medium collected from cells exposed to all three UV bands or in

medium collected from irradiated cells co-incubated with unexposed

cells. The level of IL-6 in the medium obtained in experiments with co-

incubation was higher than in medium collected from irradiated cellsincubated alone (Fig. 11); thus IL-6 must also be generated by the

nonirradiated cells, especially in UVB and UVC experiments. Possibly

senescent cells participate because an increase in IL-6 observed in the

co-incubation system after UVC and UVB exposure was associated

with senescence induction in bystander cells (compare Fig. 5 vs

Fig. 11b).

Elevation of IL-6 and IL-8 in conditioned medium has been

observed for various cancer cells after ionizing radiation, and their

action was associated with different proles of bystander cell

survival (a decrease or an increase) depending on the cell line[42].

This suggests that the generation, as well as the reception, of these

cytokines is highly cell-type specic.

The prole of secretion of IL-8 with its decrease to below

control after irradiation of NHDF broblasts by UVB and UVC was

0

200

400

600

800

15 min 3 h 6 h 12 h 24 h

IL-6(pg/ml)

Ct

UVA

UVB

UVC

15 min 3 h 6 h 12 h 24 h

Ct + BY

UVA + BY

UVB + BY

UVC + BY

15 min 3 h 6 h 12 h 24 h

Incubation time

Ct + BY

UVA + BY

UVB + BY

UVC + BY

0

300

600

900

1200

1500

15 min 3 h 6 h 12 h 24 h

Incubation time

Ct

UVA

UVB

UVC

IL-8(pg/ml)

Fig. 11. Concentration of IL-6 (top) and IL-8 (bottom) in culture medium collected from control and UV-exposed cells. (a, c) Medium from cells incubated without neighbors

in inserts; (b, d) medium from cells co-incubated with cells in inserts. UV doses were the same as those shown in Fig. 4. Data are means7SD from two independent

experiments performed in duplicate. A signicant increase in IL-6 concentration relative to control was measured between 15 min and 6 h in both systems (po0.05,

Students t test).


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rather unexpected as this cytokine was proposed as one of the

factors participating in bystander signaling after ionizing radiation

[42] and we observed an increase in IL-8 release after UVA

irradiation (Fig. 11c). The presence of nonirradiated broblasts in

the neighborhood of irradiated ones caused inhibition of IL-8

release to below that observed in medium collected from control

cells. The doses used for UVB and UVC may not stimulate IL-8

production because its production is not simply dependent on the

dose, as was observed in the case of ionizing radiation [42].However, a decrease in IL-8 under co-incubation conditions does

not necessarily mean that this cytokine is not involved in bystan-

der signaling; it may also suggest an induction of inhibitory factors

in paracrine regulation mechanisms or maybe its binding by

receptors on bystander cells. Expression of IL-8 is regulated by

the NF-B signaling pathway and reactive oxygen species arenecessary for activation of transcription complex [44]. Thus, it is

possible that an interplay between induction of ROS and intracel-

lular antioxidants is crucial for the observed effects.

All three UV wavelength bands induced reactive oxygen species

and signicantly increased the levels of superoxide dismutase.

Superoxide radical anions were generated at a higher level by UVC

compared with UVA and UVB radiation (Fig. 7). Other studies

suggest that superoxide radicals formed by respiratory complex I

in mitochondria, which can be converted to hydrogen peroxide by

superoxide dismutase, and then further to water by catalase, or to

the hydroxyl radical in the presence of transition metals[4547],

are involved in bystander effects and genomic instability because

catalase inhibits these effects [7,11]. After exposure of NHDF

broblasts to UVA, the rapid increase in the frequency of apoptosis

was paralleled by increases in ROS and superoxide radicals, which

activated SOD enzyme (compareFig. 4vs Figs. 6, 7, and 10). These

increases were not accompanied by an increase in mitochondrial

membrane potential but rather by a decrease (Fig. 8a), which

reects mitochondrial membrane depolarization. This, however,

did not appear to reect irreversible damage to mitochondria

because after 24 h apoptosis had returned to the control level and

cell survival was normal. After exposure of broblasts to UVB, a

large increase in apoptosis at 24 h was correlated with an increase

in superoxide radicals (Fig. 4b vsFig. 7b) and a slight decrease in

membrane potential (Fig. 8b). However, in some bystander cells

(UVA and UVB) and in UVC-exposed cells a signicant increase in

mitochondrial membrane potential (mit) was noticed. Different

cells may respond to UV radiation in various ways. Reduction of

the mitochondrial membrane potential in bystander cells has been

associated with increased production of ROS in human melano-

cytes after exposure to UVA, but not UVB [48]. Also, human

keratinocytes responded, by increasing ROS and reducing mito-

chondrial membrane potential, to bystander signals contained in

medium collected from cells exposed to rays[49]. In both cases areduction in mitwas associated with an inux of calcium ions into

the cells. In another study, ionizing-radiation-induced increase in

ROS in lymphoma cells was accompanied by an increase inmitochondrial membrane potential and apoptosis [50]. Further-

more, impairment of mitochondrial membrane may appear as

initial membrane hyperpolarization (increase in mit) followed by

depolarization (decrease in mit)[51],which probably is the case

in bystander cells in our UVA and UVB experiments (Fig. 8).

We can also assume that different responses of the mitochondrial

membrane to stress induced by UV radiation result from different

levels and/or natures of signaling molecules at a given time

generated by different bands of UV. However, more studies are

required to investigate the underlying mechanisms.

A highly signicant increase in the level of ROS in UVC-

irradiated and bystander cells appearing by 12 h was accompanied

by an increase in mitochondrial membrane potential. At the

same time, a signicant decrease in nitric oxide levels occurred

in UVC-irradiated and bystander cells. These changes (reduction in

NO production, increase in ROS, and increase in mit) seen at 12 h

might be associated with cell cycle inhibition, especially as an

arrest in G1 was seen after UVC radiation (G2/M:G10,18 at 12 h

vs 0.5 at the start of treatment, data not presented). However, it

appears that NO plays a minor role in the action of all UV radiation

bands and in bystander effects in our experimental system,

agreeing with studies of effects of UVB in human keratinocytes

in which NO levels were only weakly associated with apoptosisand mitochondrial dysfunction[52].

Conclusions

In conclusion, our results demonstrate that all three wave-

length bands of UV radiation caused a reduced survival and an

increased frequency of apoptosis in nonirradiated bystander

human dermal broblasts co-incubated with irradiatedbroblasts,

although with varying efciency and kinetics. In addition, UVR

induced premature senescence, in particular in UVC-exposed and

bystander cells. Our results are consistent with the idea that these

bystander effects are caused by an increase in the level of cellular

ROS in irradiated cells. Increased secretion of interleukin-6 sug-

gests its role as a molecular bystander signal released by irradiatedcells, but mutual signaling between irradiated and bystander cells

modulates this secretion. Further studies are required to under-

stand the nature of the mediators of these UV-induced bystander

effects, but nonetheless the present results showing that all three

bands efciently induced a damaging bystander effect through

generation of ROS and proinammatory cytokine suggest that UVR

carries a potential health risk not only due to direct mechanisms,

but also due to the bystander phenomenon.

Acknowledgments

This work was supported by Grants NN 518 497 639 from the

Polish Ministry of Science and Higher Education and DEC-2012/05/B/ST6/03472 from the National Center of Science. Ronald Hancock

(Laval University, Laval, QC, Canada) is acknowledged for critically

reading and editing the English of the manuscript.

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