ORIGINAL PAPER

Dose-rate effects of protons on in vivo activation of nuclearfactor-kappa B and cytokines in mouse bone marrow cells

Kanokporn Noy Rithidech • Paiboon Reungpatthanaphong •

Louise Honikel • Adam Rusek • Sanford R. Simon

Received: 9 October 2009 / Accepted: 11 May 2010 / Published online: 28 May 2010

� Springer-Verlag 2010

Abstract The objective of this study was to determine the

kinetics of nuclear factor-kappa B (NF-jB) activation and

cytokine expression in bone marrow (BM) cells of exposed

mice as a function of the dose rate of protons. The cytokines

included in this study are pro-inflammatory [i.e., tumor

necrosis factor-alpha (TNF-a), interleukin-1beta (IL-1b),and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and

IL-10). We gave male BALB/cJ mice a whole-body expo-

sure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons,

delivered at 5 or 10 mGy min-1, the dose and dose rates

found during solar particle events in space. As a reference

radiation, groups of mice were exposed to 0 (sham-controls)

or 1 Gy of 137Cs c rays (10 mGy min-1). After irradiation,

BM cells were collected at 1.5, 3, 24 h, and 1 month for

analyses (five mice per treatment group per harvest time).

The results indicated that the in vivo time course of effects

induced by a single dose of 1 Gy of 100 MeV protons or137Cs c rays, delivered at 10 mGy min-1, was similar.

Although statistically significant levels of NF-jB activation

and pro-inflammatory cytokines in BM cells of exposed

mice when compared to those in the corresponding sham

controls (Student’s t-test, p\ 0.05 or\0.01) were induced

by either dose rate, these levels varied over time for each

protein. Further, only a dose rate of 5 mGy min-1 induced

significant levels of anti-inflammatory cytokines. The

results indicate dose-rate effects of protons.

Introduction

Galactic cosmic rays (GCRs) and solar particle events

(SPEs) are the greatest sources of radiation exposure to

astronauts venturing beyond Earth. Hence, it is vitally

important to protect astronauts in space environments. This

will require improvement in our knowledge of radiation-

induced cellular and molecular changes that may impact

the health of astronauts. Moreover, for research on radia-

tion-induced genetic damage to be useful for risk assess-

ment, it must be established in in vivo systems, especially

in cells that are known to be at risk for health problems

associated with radiation exposure such as bone marrow

(BM) cells, the known target cell for radiation-induced

leukemia. While the biological effects of GCR particles, in

particular those with large mass (Z) and high energy (E)

(known as HZE particles, such as 56Fe ions) have been

investigated extensively (Durante and Cucinotta 2008),

little is known about the biological effects of SPE

components.

The SPEs are unpredictable in terms of the time of

occurrence and the size of the event (Cucinotta 1999;

Kim et al. 2009). Further, SPEs may last for several hours

up to a few days (Shea and Smart 1994). Exposure to

radiation during large SPEs can be life threatening if the

This manuscript is based on a contribution given at the ‘‘Heavy Ions

in Therapy and Space Symposium 2009,’’ July 6–10, 2009, Cologne,

Germany.

K. N. Rithidech (&) � P. Reungpatthanaphong � L. Honikel �S. R. Simon

Pathology Department, Stony Brook University,

Stony Brook, NY 11794-8691, USA

e-mail: krithidech@notes.sunysb.edu

Present Address:P. Reungpatthanaphong

Radio-Isotope Department, Faculty of Sciences,

Kasetsart University, Chatuchak, Bangkok 10900, Thailand

A. Rusek

Accelerator Department, NASA Research Laboratory,

Brookhaven National Laboratory, Upton, NY 11973-5000, USA

123

Radiat Environ Biophys (2010) 49:405–419

DOI 10.1007/s00411-010-0295-z

astronauts are inadequately protected (Cucinotta 1999; Hu

et al. 2009). Various organ dose rates (0–1 Gy h-1)

(Parsons and Townsend 2000) and doses (0–2 Gy)

(Wilson et al. 2006) of radiation from SPEs have been

estimated. The majority of ions produced during SPEs are

energetic protons ([30 MeV up to hundred and occa-

sionally thousands of MeV), followed by helium nuclei

(Cleghorn et al. 2004; Zapp et al. 1999). The flux of these

energetic protons drops rapidly with increasing energy

(Smart and Shea 1989). A large SPE produces very

intense charged particles composed almost entirely of

protons below 150 MeV. For these reasons, the focus of

this study was on the biological effects of 100 MeV

protons, which are one of the most abundant types of

protons induced during SPEs (Simpson 1983; Smart and

Shea 2002) making them the ions of interest for space-

radiation risk assessment. Further, information on bio-

logical effects of 100 MeV protons is currently lacking.

In contrast, using varying endpoints (e.g., micronu-

cleus assay and cell transformation), a few in vitro or in

vivo studies have been conducted to examine biological

effects of protons with different energies, i.e., 55, 200,

232, or 250 MeV, delivered at different dose rates,

ranging from 20 mGy min-1, 7, and 1 Gy min-1 (Chang

et al. 2005; Elmore et al. 2005; Green et al. 2001;

Gueulette et al. 2000). The focus of this study was on

the dose-rate effects of 100 MeV protons on the in vivo

activation of NF-jB and the expression of NF-jB-relatedproteins. As mentioned above, protons with varying

energies below 150 MeV are produced during SPEs;

thus, the data presented here are part of a study series to

investigate the in vivo biological effects of protons with

varying energies below 150 MeV, i.e., 30, 50, and 100

MeV.

In the past, it was generally assumed that protons induce

the same level of biological effects as those produced by X

and c rays. However, results from an in vitro study indi-

cated that 250 MeV protons induced higher levels of

damage (e.g., micronuclei, cell killing) in exposed rat

thyroid cells than in cells exposed to 60Co c rays, while thecell cycle phase distribution was not statistically different

(Green et al. 2001). Moreover, an in silico study indicated

that 1 GeV protons produce a higher level of clustered

DNA damage (presumably leading to subsequent double-

strand breaks) when compared to X or c rays (Hada and

Sutherland 2006). This important finding suggests that

health risks from exposure to protons (*1 GeV) may be

higher than previously thought. Further, it also shows that

protons with varying energies (most likely with diverse

LET) induce different levels of biological effects. More-

over, these results were in accordance with previously

observed phenomenon on LET-dependent biological

effects of radiation (Barendsen 1968, 1994).

Dose-rate effects of c or X rays, but not protons, have

been studied extensively. It has been well established, both

in in vivo and in vitro systems, that the clastogenic,

mutagenic, and carcinogenic activities of c or X rays

decrease for a given dose as the dose rate is decreased

(BEIR 2005; Boreham et al. 2000; Cornforth et al. 2002;

Elmore et al. 2006; Geard and Chen 1990; Kato et al. 2006;

Kumar et al. 2006; Little and Muirhead 1997; Loucas et al.

2004; Preston et al. 2002). Currently, there is no informa-

tion on the dose/dose-rate effects of 100 MeV protons

which are more abundant than 1 GeV protons during SPEs.

To provide important information directly related to the

NASA mission, we conducted experiments describing the

differences in dose-rate effects of 1 Gy of 100 MeV pro-

tons. Groups of mice were exposed to 100 MeV protons of

one selected moderate dose (1 Gy) delivered at two dif-

ferent dose rates (5 or 10 mGy min-1). As a reference

radiation, groups of mice were exposed to 0 or 1 Gy of137Cs c rays (delivered at 10 mGy min-1). Only one dose

rate of 137Cs c rays was used because there is abundant

information on dose/dose-rate effects of 137Cs c rays.

Further, the lowest dose rate possible from the c-irradiator(located in the Biology Department, Brookhaven National

Laboratory, Upton, New York) used in this study is

10 mGy min-1. Mice included in this study were also part

of a study series conducted to examine the extent and the

mechanisms involved in in vivo induction of genomic

instability (expressed as late occurring chromosome insta-

bility) by 100 MeV protons in which BM cells were col-

lected from groups of mice for analysis at different times

post-exposure. The scope of this study was to determine the

kinetics of NF-jB activation and the expression of selected

NF-jB target proteins known to be involved in inflam-

matory response, i.e., pro-inflammatory cytokines (TNF-a,IL-1b, and IL-6) and anti-inflammatory cytokines (IL-4

and IL-10) in BM cells of exposed mice.

An inflammatory process is the first cellular defense

mechanism in response to stress-inducing agents, including

radiation, in which several pro- and anti-inflammatory

cytokines are produced. NF-jB is a key transcription factor

involved in regulating the expression of such cytokines

(Baldwin 1996; Ghosh et al. 1998). An imbalance between

levels of pro-inflammatory and anti-inflammatory cyto-

kines at early and late times post-irradiation may contribute

to perpetuating inflammation. If inflammation or the pro-

duction of pro-inflammatory cytokines persists (chronic

inflammation), it becomes detrimental to exposed cells or

tissues. Further, NF-jB activation is also an important

contributor to cell survival and protection from apoptosis

following exposure to DNA-damaging agents in vitro (Liu

et al. 1996; Weichselbaum et al. 1994) and in vivo (Wang

et al. 2004). NF-jB is a member of the ubiquitously

expressed family of the Rel-related transcription factors

406 Radiat Environ Biophys (2010) 49:405–419

123

(Verma et al. 1995). These are p65 (RelA), p68 (RelB), p50

(NF-jB1), p52 (NF-jB2), and c-Rel. NF-jB proteins

interact to form homo- or heterodimers that bind DNA with

different specificities. The most prevalent and well studied

is NF-jB/p65. Hence, the activation of NF-jB/p65 is the

focus of this study.

The results from several in vitro studies showed that

low LET radiation (at varying dose and dose-rate levels)

induces NF-jB activation in different cell types such as

human lymphoblastoid cells (Mohan and Meltz 1994;

Prasad et al. 1994), human KG-I myeloid leukemia cells

(Brach et al. 1991), human glioma cell lines (Basu et al.

1998), human B-cells (Rho et al. 2005), or mouse skin

epithelial cells (Fan et al. 2007). Depending on the cell

types, elevation of activated NF-jB was detected at early

time points (1–8-h post-irradiation), then the levels of

activated NF-jB declined toward the level detected in

non-irradiated cells within 24 h. In contrast, information

on radiation-induced NF-jB activation in vivo is scant

(Rithidech et al. 2005; Zhou et al. 1999a, 2001). Like-

wise, there is little information on NF-jB activation

induced by space radiation and the existing knowledge is

derived from in vitro studies only. These include NF-jBactivation in human monocytes by 1 GeV/n 56Fe ions,

delivered at the dose rate of 1 Gy min-1 (Natarajan et al.

2002) and in human embryonic kidney (293/HEK) cells

by 95 MeV/n 36Ar ions (Baumstark-Khan et al. 2005;

Hellweg et al. 2005). To date, there is no information,

either in vitro or in vivo, on the activation of NF-jB as

well as the expression of NF-jB-targeted cytokines by

100 MeV protons. This study is the first to study the dose-

rate effects of 100 MeV protons on in vivo activation of

NF-jB and cytokines in a total cell population of the BM

using a mouse model.

Materials and methods

Animals

All male BALB/cJ mice were purchased from the Jackson

Laboratory (Bar Harbor, ME) and were delivered directly

from the vendor to Brookhaven National Laboratory

(BNL), Upton, New York, where irradiation took place.

They were allowed 2 weeks to acclimate prior to their

irradiation (at 10–12 weeks old, with an average body

weight of 25 g). They were housed in a facility approved

by the Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC) at BNL. The animal

rooms were maintained with the light cycle of 12 h light/

12 h dark, 21 ± 2�C, with 10-15 hourly cycles of fresh air

and a relative humidity of 50 ± 10%. The protocols for

animal housing and care, including experimental design of

the study, were approved by both the BNL and the Stony

Brook University (SBU) Institutional Animal Care and Use

Committee (IACUC).

Irradiation of mice

For 100 MeV proton-irradiation, the exposure was done at

the NASA Research Laboratory (NSRL) located at BNL,

Upton, New York. Mice were exposed whole body to the

average total-body doses of 0 or 1 Gy, delivered at the dose

rates of 5 or 10 mGy min-1 by a 20 9 20 cm beam. Mice

exposed to 0 Gy served as sham controls. As previously

mentioned, the selected dose and dose rates of 100 MeV

protons are comparable to those found in space. Further,

only 1 Gy of protons was used to compare with 0 Gy

(sham-control) because the focus of this study was on the

influence of dose rate, not the dose–response effect. For

each dose rate, a total of 40 mice were used. The dose-

average LET at the sample position was 0.7 keV/lm. The

direction of the 100 MeV protons to the target was hori-

zontal. The uniformity of the beam was measured to ensure

even dose distribution across the field of exposure. Further,

the exposure chambers were rotated 180 degrees at 10-min

intervals. Exposure procedures described previously

(Rithidech et al. 2005) were followed. Groups of mice that

were used for sample collection at 1-month post-irradiation

(10 mice for each dose of each dose rate of 100 MeV

protons) were transported back to SBU in a climate-

controlled vehicle within 1-week post-exposure. The

animal facility of SBU is also approved by the Association

for Assessment and Accreditation of Laboratory Animal

Care (AAALAC), with the same light cycle (12 h light/

12 h dark), temperature (21 ± 2�C), 10–15 hourly cycles

of fresh air, and relative humidity (50 ± 10%) as those

of BNL. Sample collections at 1.5, 3, and 24 h were

performed at the BNL Animal Facility.

For c-irradiation (a reference radiation, LET = 0.662

keV/lm), two groups of 20 mice in each were given a

whole body dose of 0 and 1 Gy of 137Cs c rays (at the doserate of 10 mGy min-1) using a gamma-irradiator located at

the Biology Department, BNL. Exposure procedures used

for 100 MeV protons were followed. Further, the direction

of c rays to the target was also horizontal. The schedule of

sample collections was the same as in mice exposed to

100 MeV protons.

Collection of bone marrow cells

At each harvest time for each dose rate, we collected BM

cells from each mouse (five mice per radiation dose) by

flushing both femurs and tibiae with 10 mL of McCoy’s

5A medium. As mentioned in the ‘‘Introduction’’ section,

the measurement of NF-jB activation presented in this

Radiat Environ Biophys (2010) 49:405–419 407

123

study is part of a study series examining the induction of

genomic instability (expressed as late occurring chromo-

somal damage). Consequently, the majority of BM cells

collected from each mouse were used in molecular cyto-

genetics for the detection of chromosomal damage.

Availability of BM cells used for the measurements of

NF-jB activation and levels of cytokines from each

mouse was therefore limited. Approximately 3 9 106

cells per mouse were available for the measurement of

NF-jB or cytokines.

Measurement of NF-jB activation

We used the enzyme-linked immunosorbent assay (ELISA)

NF-jB/p65 kits from Active Motif North America Inc.

(Carlsbad, CA) for measuring NF-jB activation in nuclear

extracts obtained from BM cells (approximately 3 9 106

cells) collected from each mouse at different times post-

irradiation (Rithidech et al. 2005). Nuclear extract kits

(Active Motif, Carlsbad, CA) were used for obtaining

nuclear lysates. Activation of NF-jB was measured using a

microplate spectrophotometer (Molecular Devices, Sun-

nyvale, CA) at 450 nm. It also should be noted that the data

obtained from microplate spectrophotometer readings rep-

resented the corrected values of the rate of tetra-methyl

benzidine conversion (expressed as mOD/minute) in actual

samples (including sham controls) after subtraction of the

substrate conversion rates in reagent blank samples. The

assay was performed in duplicate wells for each BM

sample.

Measurement levels of TNF-a, IL-1b, IL-6, IL-4,and IL-10

We measured the expression levels of these selected

cytokines involved in inflammatory response and known to

be regulated by NF-jB activation in BM cells (the cyto-

solic fraction) using the specific ELISA kits for each

cytokine from Biosource (Invitrogen, Carlsbad, CA).

Commercially available extraction kits (Active Motif,

Carlsbad, CA) were used for obtaining the cytosolic frac-

tion of BM cells. The method of extraction suggested by

the manufacturer was followed. Serial dilutions of known

concentrations of each cytokine were used to create a

standard curve for each of the cytokines included in the

study. The concentrations of the cytokines in BM samples

were calculated from their respective standard curves. The

levels of each cytokine were measured using a microplate

spectrophotometer (Molecular Devices, Sunnyvale, CA) at

450 nm. Similar to the measurements of activated NF-jB,the assay for each cytokine was performed in duplicate

wells for each BM sample.

Statistical analysis

We expressed NF-jB and cytokine data as mean ± SEM

and performed statistical analysis with the Student’s t-test.

Differences in these values between the exposed group and

the corresponding sham-control mice were considered

statistically significant at p-values\ 0.05.

Results

The summaries of the results are shown in Figs. 1, 2, 3, 4,

5, and 6. Each data point shown in each figure represents

mean values from 10 measurements (5 mice per group, 2

measurements per mouse) ± standard error of the mean

(SE). Additionally, the fold changes in levels of expression

of each protein in BM cells after exposure of mice to

radiation relative to the levels in their corresponding sham

controls at each time point were also presented in the fig-

ures adjacent to the relative amount of protein for each

radiation treatment protocol. The details of each measure-

ment are presented below.

NF-jB activation

The level of activated NF-jB in BM cells of male BALB/

cJ mice collected at 1.5 h after exposure to 1 Gy of

100 MeV protons delivered at 5 mGy min-1 did not differ

from that in sham controls (Fig. 1a). However, highly

significant levels (p\ 0.05) of activated NF-jB in BM

cells of exposed mice were found at 3- and 24-h post-

irradiation, and persisted up to 1-month post-exposure

(p\ 0.01). Such increases were 1.3-, 1.8-, and 3.8-fold

higher than those in their corresponding sham control

levels at 3-h, 24-h, and 1-month post-irradiation, respec-

tively (Fig. 1a.1).

In contrast to the exposure at the dose rate of

5 mGy min-1, the level of activated NF-jB in BM cells

collected at 1.5 h after exposure of male BALB/cJ mice to

1 Gy of either 100 MeV protons delivered at 10 mGy

min-1 or 137Cs c rays was significantly increased

(p\0.01) above that in the sham control samples (Fig. 1b, c).

Subsequently, at 3- and 24-h post-exposure, there was a

reduction in the levels of activated NF-jB in BM cells of

both sham control and exposed mice. However, the levels

of activated NF-jB in BM cells of exposed mice were still

higher (but not significantly) than those in their corre-

sponding sham controls. At late time (1 month) post-

exposure, a significant increase (p\ 0.05) in the level of

activated NF-jB was detected in BM cells of exposed

mice when compared to that in the BM cells of sham

controls. The results also indicate that a single dose of

408 Radiat Environ Biophys (2010) 49:405–419

123

1 Gy of 100 MeV protons exhibited a similar pattern of

effects to those induced by a single dose of 1 Gy of 137Cs crays, delivered at the same dose rate (10 mGy min-1). It

also should be noted that the levels of NF-jB activation in

BM cells of proton-exposed mice relative to those in their

corresponding sham controls were slightly higher than

those in the c-exposed mice compared to their corre-

sponding sham controls (Fig. 1b.1, c.1).

TNF-a

At 1.5 and 24 h (but not at 3 h) after exposure, the levels of

TNF-a in BM cells were significantly increased (p\ 0.01)

above those in their corresponding sham controls after

exposure of male BALB/cJ mice to 1.0 Gy of 100 MeV

protons delivered at 5 mGy min-1 (Fig. 2a). Further, the

highly significant level of TNF-a in BM cells of exposed

Fig. 1 Time course of NF-jB/p65 activation, measured by the

ELISA Trans-AM kits (Active

Motif North America Inc.,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in levels

of activated NF-jB/p65 induced

by each exposure regimen

relative to the corresponding

sham-control levels are shown

in a.1, b.1, and c.1, respectively.Significant differences in the

levels of activated NF-jB/p65are shown at *

Radiat Environ Biophys (2010) 49:405–419 409

123

mice was persistent up to 1-month post-exposure

(p\ 0.01). However, the TNF-a levels were only about

1.5-fold increased in relation to those in their correspond-

ing sham controls at each harvest time (Fig. 2a.1).

Similar to the effects of 5 mGy min-1, the levels of

TNF-a in BM cells were significantly increased (p\ 0.01)

above those in the sham control samples collected at 1 and

24 h (but not at 3 h) after exposure of male BALB/cJ mice

to 1.0 Gy of either 100 MeV protons delivered at

10 mGy min-1 (Fig. 2b) or 137Cs c rays (Fig. 2c).

Likewise, a significant elevation of TNF-a was found in

BM cells of mice exposed to either 1 Gy of 100 MeV

protons or 137Cs c rays at 1-month post-exposure

(p\ 0.01). The TNF-a levels in BM cells of exposed mice

showed 4.8-, 3.1-, and 1.3-fold increases relative to those

observed in their corresponding sham controls at 1.5-h,

24-h, and 1-month post-exposure to protons, respectively

(Fig. 2b.1). With respect to c-irradiation, although the

pattern of TNF-a expression as a function of time post-

exposure was similar to that induced by 100 MeV protons,

Fig. 2 Time course of TNF-aexpression, measured by the

ELISA TNF-a kits (Invitrogen,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in the

expression levels of TNF-ainduced by each exposure

regimen relative to the

corresponding sham-control

levels are shown in a.1, b.1, andc.1, respectively. Significantdifferences in the levels of TNF-aexpression are shown at *

410 Radiat Environ Biophys (2010) 49:405–419

123

the increases in TNF-a levels in exposed mice relative to

the corresponding sham control levels were 1.5-, 3.2-, and

1.6-fold at 1.5-h, 24-h, and 1-month-post-exposure,

respectively (Fig. 2c.1).

IL-1b

At 1.5-h, 24-h, and 1-month after exposure, the levels of

IL-1b in BM cells were significantly increased (p\ 0.01)

above those in their corresponding sham controls after

exposure of male BALB/cJ mice to 1.0 Gy of 100 MeV

protons delivered at 5 mGy min-1 (Fig. 3a). The levels of

IL-1b detected in BM cells of proton-exposed mice were

1.4-, 1.7-, and 1.3-fold increased relative to those in the

corresponding sham control levels at 1.5-h, 24-h, and

1-month post-exposure, respectively (Fig. 3a.1). No dif-

ference in the IL-1b level in BM cells of exposed and sham

control mice was found at 3-h post-exposure.

Fig. 3 Time course of IL-1bexpression, measured by the

ELISA IL-1b kits (Invitrogen,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in the

expression levels of IL-1binduced by each exposure

regimen relative to the

corresponding sham-control

levels are shown in a.1, b.1, andc.1, respectively. Significantdifferences in the levels of

IL-1b expression are shown at *

Radiat Environ Biophys (2010) 49:405–419 411

123

In contrast to the dose rate of 5 mGy min-1, the IL-1blevels in BM cells collected at 24 h after exposure of mice

to either 100 MeV protons delivered at 10 mGy min-1

(Fig. 3b) or 137Cs c rays (Fig. 3c) did not differ from those

of the corresponding sham control levels. However, sig-

nificant levels of IL-1b were detected in BM cells collected

at 1.5-h, 3-h, and 1-month after exposure of BABL/cJ mice

to either type of radiation, when compared to those in their

corresponding sham controls, with p-values ranging from

p\ 0.01 to \0.05. Figure 3b.1 and c.1 show the fold

changes in IL-1b levels detected in BM cells collected

from irradiated mice relative to those detected in the cor-

responding sham controls.

Il-6

After irradiation with 100 MeV protons at a dose rate of

5 mGy min-1, the expression levels of Il-6 in BM cells

Fig. 4 Time course of IL-6

expression, measured by the

ELISA IL-6 kits (Invitrogen,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in the

expression levels of IL-6

induced by each exposure

regimen relative to the

corresponding sham-control

levels are shown in a.1, b.1, andc.1, respectively. Significantdifferences in the levels of IL-6

expression are shown at *

412 Radiat Environ Biophys (2010) 49:405–419

123

collected from exposed mice were statistically higher than

those in the sham controls at 1.5-h (p\ 0.05), 24-h

(p\ 0.1), and 1-month (p\ 0.05) post-irradiation

(Fig. 4a). The levels of IL-6 in BM cells of exposed mice

were 1.4-, 1.7-, and 1.3-fold of those detected in the cor-

responding sham control levels (Fig. 4a.1). At 3 h after

exposure, however, IL-6 levels did not differ between

irradiated and sham control mice.

Expression levels of Il-6were highly significant inBMcells

of mice exposed to 1 Gy of 100 MeV protons delivered at

10 mGy min-1 (p\0.05, Fig. 4b) or 137Cs c rays (p\0.01,

Fig. 4c) only at 3-h post-exposure, when compared to

those of their corresponding sham controls. The highest

increase in IL-6 level related to that of the sham control

was found at 3-h post-irradiation, i.e., 1.8-fold for protons

and 2.3-fold for 137Cs c rays (Fig. 4b.1, c.1, respectively).

Fig. 5 Time course of IL-4

expression, measured by the

ELISA IL-4 kits (Invitrogen,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in the

expression levels of IL-4

induced by each exposure

regimen relative to the

corresponding sham-control

levels are shown in a.1, b.1, andc.1, respectively. Significantdifferences in the levels of IL-4

expression are shown at *

Radiat Environ Biophys (2010) 49:405–419 413

123

IL-4

The expression levels of IL-4 were significantly elevated in

BM cells collected at 1.5 h (p\ 0.05) and 24 h (p\ 0.01)

from mice exposed to 100 MeV protons at 5 mGy min-1

(Fig. 5a), when compared to their corresponding sham

controls. Such increases were 1.25- and 1.4-fold of the

corresponding sham control levels at 1.5 and 24 h,

respectively (Fig. 5a.1). At 3-h post-irradiation, the level of

IL-4 in BM cells of exposed mice was lower than that

detected in the sham control group. Further, there were no

differences in expression levels of IL-4 in BM cells of

exposed and sham control mice collected at 1-month post-

irradiation.

In contrast to the irradiation at the dose rate of

5 mGy min-1, there was a significant suppression of IL-4

levels in BM cells collected at 1.5 h after exposure of mice

to either 100 MeV protons delivered at 10 mGy min-1

Fig. 6 Time course of IL-10

expression, measured by the

ELISA IL-10 kits (Invitrogen,

Carlsbad, CA), in BM cells

collected from male BALB/cJ

mice following in vivo exposure

to 0 or 1 Gy of 100 MeV

protons delivered at

5 mGy min-1 (a) or at10 mGy min-1 (b). c Shows theeffects of 1 Gy of 137Cs c rays

(a reference radiation) delivered

at 10 mGy min-1. Each point

represents mean ± SE of five

mice (two measurements per

mouse). Fold changes in the

expression levels of IL-10

induced by each exposure

regimen relative to the

corresponding sham-control

levels are shown in a.1, b.1, andc.1, respectively. Significantdifferences in the levels of

IL-10 expression are shown at *

414 Radiat Environ Biophys (2010) 49:405–419

123

(p\ 0.01, Fig. 5b, b.1) or 137Cs c rays (p\ 0.01, Fig. 5c,

c.1). Apparently, IL-4 levels in the exposed groups

remained lower than those in their corresponding sham

controls throughout the study (but not significant), although

there seemed to be some recovery of IL-4 production.

IL-10

Similar to the expression levels of IL-4, there were highly

significant levels of IL-10 in BM cells collected at 1.5 h

(p\ 0.05) and 24 h after exposure of mice (p\ 0.01) to

100 MeV protons at 5 mGy min-1 (Fig. 6a). There were

1.2- and 1.35-fold increases in IL-10 levels in exposed

groups relative to their corresponding sham controls at 1.5

and 24 h, respectively (Fig. 6a.1). At 3-h post-irradiation,

the level of IL-10 in BM cells of exposed mice was lower

than that detected in the sham control group. Further, the

IL-10 level in BM cells collected at 1 month from exposed

mice did not differ from that detected in their corre-

sponding sham controls.

There was a significant suppression of IL-10 levels in

BM cells collected at 1.5 h after exposure of mice to 1 Gy

of either 100 MeV protons delivered at 10 mGy min-1

(Fig. 6b) or 137Cs c rays (Fig. 6c). Subsequently, the pro-

duction of IL-10 was gradually increased in exposed mice,

but the levels of IL-10 remained lower than those detected

in the sham control groups (Fig. 6b, b.1, c, c.1).

Discussion

Our in vivo data demonstrated the impact of the dose rate

of 100 MeV protons on the pattern of NF-jB activation (as

well as the subsequent expression of proteins involved in

inflammatory response, i.e., cytokines known to be regu-

lated by activated NF-jB) in the total population of BM

cells of exposed mice. The cytokines included in this study

are pro-inflammatory cytokines (TNF-a, IL-1b, IL-6) andanti-inflammatory cytokines (IL-4 and IL-10). It is known

that the anti-inflammatory cytokines are necessary for

controlling and down-regulating inflammation (possibly by

counterbalancing the elevation of the pro-inflammatory

cytokines) and for maintaining homeostasis. In contrast,

the pro-inflammatory cytokines are primarily responsible

for initiating an effective inflammatory process. Our data

also demonstrated that a single dose of 1 Gy of 100 MeV

protons exhibited similar effects to those induced by a

single dose of 1 Gy of 137Cs c rays, delivered at the same

dose rate (10 mGy min-1). It also should be noted that

such a conclusion cannot be drawn between a dose rate of

5 mGy min-1 of 100 MeV protons and 137Cs c rays, sincethere were no data for the 5 mGy min-1 of 137Cs c rays forcomparison. However, it has been reported that increased

levels of pro-inflammatory cytokines were found with

increasing dose rates of 137Cs c rays (Ross et al. 1997).

We recognize that there are many other NF-jB-medi-

ated cytokines and that the production of these cytokines is

controlled by complex feedback mechanisms (Linard et al.

2003; Schreiber et al. 1998). The cytokines TNF-a, IL-1b,IL-4, IL-6, and IL-10 were selected for study because their

expressions, at the gene level, were elevated in human

mononuclear cells obtained from healthy adult individuals

who lived near the Chernobyl Nuclear Power Plant and

were chronically exposed to low-dose radiation ranging

from 0.18 to 49 mSv (Albanese et al. 2007). Further, these

cytokines were detected in the spleen or macrophages of

mice exposed to varying doses of radiation (ranging from

0.05 to 7.0 Gy) (Chang et al. 2000; Hosoi et al. 2001; Liu

et al. 2003). Additionally, these cytokines were detected in

the ileal muscular layer when a single dose of 10 Gy of60Co c rays (at a dose rate of 5 mGy min-1) was given to

the abdomen of male Wistar rats (Linard et al. 2004), or in

the bronchiolar epithelial cells when a single dose of 12 Gy

of 10 MV photons from a linear accelerator was given to

the thoracic region of C57BL/6J mice (Rube et al. 2005).

Exposure to 1 Gy of 100 MeV protons or 137Cs c rays

(delivered at 10 mGy min-1) rapidly activated NF-jB.Using this dose rate, a highly significant level of activated

NF-jB was detected as early as 1.5-h post-exposure.

Subsequently, there was a reduction in levels of activated

NF-jB, although higher levels (but not significant) were

found in BM cells of mice exposed to either 100 MeV

protons or 137Cs c rays, when compared to those of their

corresponding sham controls. At 1-month post-exposure,

there was another wave of a significantly high level of

activated NF-jB. In groups of mice exposed to 1 Gy of

100 MeV protons (delivered at 5 mGy min-1), however,

NF-jB activation was gradually increased. A significant

increase in the level of activated NF-jB was first observed

at 3-h (not at 1.5 h) post-irradiation in BM cells of exposed

mice. The mechanisms potentially involved in the delay of

NF-jB activation are currently unknown.

Our studies are the first in which the measurements of

NF-jB activation are done in BM cells collected at late

time (1 month) after exposure of mice to different dose

rates of radiation, specifically those found in space. Among

the three radiation treatment protocols, the highest increase

in the level of activated NF-jB was found in BM cells

collected at 1 month after exposure of mice to a single dose

of 1 Gy of 100 MeVn protons delivered at 5 mGy min-1

(i.e., a 3.8-fold increase when compared to the corre-

sponding control level, as shown in Fig. 1a.1, while there

were 2.2- and 1.5-fold increases in mice exposed to protons

or c rays with the dose rate of 10 mGy min-1 (Fig. 1b.1,

c.1, respectively). The finding of persistent increases in

the level of activated NF-jB at this late time (1 month)

Radiat Environ Biophys (2010) 49:405–419 415

123

post-exposure among mice exposed to either type of radi-

ation suggests that chronic inflammation might occur and

warrants further investigation. As mentioned above, the

NF-jB-mediated cytokine production is a complex system

leading to a loop of feedback mechanism. It has been

shown that pro-inflammatory cytokines [such as TNF-alpha

(Pikarsky et al. 2004; Todorov et al. 2004), or IL-1b(Catley et al. 2003; Lancel et al. 2008)] can activate NF-

jB. Hence, the persistence of significantly increased NF-

jB activation detected at 1-month post-irradiation may

reflect the feedback mechanisms induced by these cyto-

kines. Thus, the feedback loop can further enhance the

inflammation process. Whether or not the persistence of

NF-jB found in this study will be linked to the induction of

genomic instability (assessed by the presence of late

occurring chromosomal damage) in BM cells of exposed

mice remains unclear. Studies directed toward the deter-

mination of such an association are being investigated in

our laboratory.

In the past, the in vivo studies were carried out to

determine only the level of NF-jB activation at early time

(\24-h post-irradiation). Further, the earlier in vivo studies

used low LET radiation and high dose rates (Rithidech

et al. 2005; Zhou et al. 1999a). In a study previously

conducted in our laboratory, we measured the levels of

NF-jB activation in BM cells only at 1 and 4 h after

exposure of BALB/cJ mice to 0, 0.05, 0.1 or 1 Gy of 137Cs

c rays (delivered at 7.5 mGy min-1) (Rithidech et al.

2005). We found that 0.1 and 1.0 Gy (but not 0.05 Gy) of137Cs c rays were capable of activating NF-jB in BM cells

collected at 1-h (but not at 4 h) post-irradiation from

BALB/cJ mice (Rithidech et al. 2005). In that study, we

also observed no increase in the frequencies of chromo-

some aberrations in the group of mice without the signal of

early-activated NF-jB (i.e., mice exposed to 0.05 Gy of137Cs c rays) at any time point included in our study up to

6-month post-irradiation (unpublished data). Overall,

although the levels of activated NF-jB were determined at

early time points after exposure only, the findings suggest a

link between radiation-induced NF-jB activation and

radiation-induced genomic instability.

Further, tissue specificity of NF-jB activation by a high

dose of 137Cs c rays (8.5 Gy) has been reported after

exposure of the BALB/c mouse (Zhou et al. 1999b). In that

study, the investigators observed the highest level of NF-

jB activation in the BM, followed by the spleen and

mesenteric lymph nodes of the BALB/c mouse. In contrast,

there was no activated NF-jB signal in liver, lung, colon,

or brain of BALB/c mice exposed to a single dose of

8.5 Gy of 137Cs c rays. This high dose of radiation induced

a rapid increase in NF-jB activation, reaching a peak

within 1-h post-irradiation. Subsequently, the level of NF-

jB activation was decreased at 2.5 h, reaching background

levels at 5 h, and was less than the background level after

5-h post-irradiation. The same group of investigators also

found high levels of activated NF-jB in intestinal epithelial

cells of the small intestine after a whole-body exposure of

mice to varying doses of 137Cs c rays (0.5–12 Gy, at the

dose rate of 2.4 Gy min-1) (Wang et al. 2004). The authors

suggested that in vivo activation of NF-jB selectively

protects the small intestine against radiation exposure.

Additionally, it was shown that NF-jB activation is

directly involved in the expression of genes coding for pro-

inflammatory cytokines (i.e., TNF-a, IL-1b, and IL-6) in

the BM tissues of exposed mice (Zhou et al. 2001).

However, there was no information on NF-jB-mediated

expression of anti-inflammatory cytokines. Since NF-jBactivation regulates the production of both pro- and anti-

inflammatory cytokines, it is imperative to determine the

levels of both types of cytokines.

Our study also is the first in that the measurements of

pro- and anti-inflammatory cytokine levels (at the protein

level) are performed in BM cells of the same individual

mouse after a whole-body exposure to 100 MeV protons

delivered at the dose rates comparable to those found in

space. Similar to NF-jB activation, the highly significant

levels of expression of pro-inflammatory cytokines TNF-aand IL1-b in BM cells of exposed mice were found at early

time post-irradiation and persisted up to 1-month post-

irradiation. This phenomenon may also reflect the feedback

loop of NF-jB-mediated cytokine production, as observed

in the lung tissue of mice after thoracic irradiation (Rube

et al. 2005). It also has previously been suggested that a

high level of pro-inflammatory cytokines may contribute to

bystander-mediated effects and to genomic instability

(Lorimore et al. 2003; Moore et al. 2005).

With respect to IL-6, our data showed that a significant

level was persistent at 1-month post-irradiation only in BM

cells collected from mice exposed to 1 Gy of 100 MeV

protons, delivered at 5 mGy min-1. When a higher dose

rate (10 mGy min-1) was used, however, the IL-6 level in

BM cells of mice exposed to either 100 MeV protons or137Cs c rays did not differ from those detected in their

corresponding sham controls. Mechanisms involved in this

difference are currently unknown. However, it should be

noted that IL-6 (unlike TNF-a or IL-1b) has both pro- and

anti-inflammatory properties (Barton 1997; Opal and

DePalo 2000). Our data demonstrated that the time course

in the fold change of expression for IL-6 found in BM cells

of mice exposed to 1 Gy of 100 MeV protons, delivered at

5 mGy min-1 (Fig. 4a.1), is similar to those of the two

anti-inflammatory cytokines selected for study, i.e., IL-4

(Fig. 5a.1) or IL-10 (Fig. 6a.1).

IL-4 and IL-10 are known to be the important anti-

inflammatory cytokines (Opal and DePalo 2000). For each

radiation treatment protocol, the patterns of expression of

416 Radiat Environ Biophys (2010) 49:405–419

123

these two anti-inflammatory cytokines are similar. Highly

significant increases in levels of both cytokines were

detected in BM cells collected from mice exposed to 1 Gy

of 100 MeV protons delivered at 5 mGy min-1 only. In

contrast, a higher dose rate (10 mGy min-1) of 100 MeV

protons or 137Cs c rays significantly suppressed the pro-

duction of these two anti-inflammatory cytokines from

1.5 h up to 1-month post-exposure. Although there was

some recovery, the levels of IL-4 or IL-10 did not reach the

levels detected in their corresponding sham controls within

1-month post-irradiation (Figs. 5b, c, 6b, and c). It has been

established that both anti-inflammatory cytokines can

inhibit the production of pro-inflammatory cytokines TNF-

a and IL-1b (Cassatella et al. 1994; Linard et al. 2003; Opal

and DePalo 2000). It is thus possible that only a small

increase in TNF-a levels (about 1.3 fold increase,

Fig. 2a.1) observed in BM cells collected from mice

exposed to 100 MeV protons (delivered at 5 mGy min-1)

reflects the counterbalance action of anti-inflammatory

cytokines. In contrast, several fold increases in TNF-alevels were detected in BM cells collected from mice

exposed to a higher dose rate (10 mGy min-1) of radiation

(Fig. 2b.1, c.1), in which the levels of IL-4 and IL-10 were

significantly suppressed. Our data, however, showed that

production of IL-1b seems to be unaffected by the pro-

duction of IL-4 and Il-10. With the radiation dose used in

our study, it is possible that the production of IL-4 and

IL-10 is inadequate resulting in insufficient control over

pro-inflammatory cytokines TNF-a or IL-1b.In summary, we have detected differential patterns of in

vivo NF-jB activation and the expression of a subset of

cytokines in mouse BM cells as a function of the dose rate

of 1 Gy of 100 MeV protons. We also have found that the

in vivo time course of effects induced by a single dose of

1 Gy of 100 MeV protons or 137Cs c rays, delivered at the

same dose rate (10 mGy min-1), was similar. Further, the

persistence of NF-jB activation and pro-inflammatory

cytokines was detected at late time (1 month) post-exposure.

Such findings suggest the occurrence of the persistence of

cytokine production, presumably due to the feedback

mechanisms. The balance or imbalance of the anti- and

pro-inflammatory cytokines after radiation exposure may

play an important role in determining the extent of the

immune response and the health outcome. Our data provide

an important initial basis for risk assessment of in vivo

exposure to protons that are abundant in space during

SPEs. However, only a subset of cytokines was studied.

There are many other NF-jB-mediated cytokines (both

pro- and anti-inflammatory cytokines) such as interferon-

alpha (IFN-a) and IL-8, as well as their receptors. To

improve our understanding of the biological effects of

protons, it will be important in subsequent studies to

determine the levels of these cytokines and their receptors

after exposure to protons. This is particularly important

because the ultimate activities of cytokines are also regu-

lated by their receptors.

Acknowledgments We thank Dr. Peter Guida and his team for

logistic support, MaryAnn Petry and her BLAF staff for their assis-

tance in animal handling. We also thank Dr. Michael Sivertz for

dosimetry support. This work was supported by the National Aero-

nautics and Space Administration (NASA) Grant #NNX07AP88G.

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