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348
International Journal of Radiation Biology, April 2012; 88(4): 348–358
© 2012 Informa UK, Ltd.
ISSN 0955-3002 print / ISSN 1362-3095 online
DOI: 10.3109/09553002.2012.654044
Correspondence: Jaroslav Pejchal, MD, PhD, Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01
Hradec Kralove, Czech Republic. Tel: � 420973 253 216. Fax: � 420495513018. E-mail: jaroslav.pejchal@seznam.cz
( Received 13 June 2011 ; revised 11 November 2011 ; accepted 12 December 2011 )
Activation of p38 MAPK and expression of TGF- β 1 in rat colon enterocytes after whole body γ -irradiation
Jaroslav Pejchal 1 , Jakub Novotný 2 , Václav Ma ř á k 2 , Jan Ö sterreicher 2 , Ale š Tich ý 2 , Ji ř ina V á vrov á 2 , Zuzana Š inkorov á 2 , Lenka Z á rybnick á 2 , Eva Novotn á 2 , Jaroslav Chl á dek 3 , Andrea Babicov á 4 , Klára Kubelkov á 1 & Kamil Ku č a 1
1 Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic, and
2 Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic
3 Department of Farmacology, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic
4 Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic
by eukaryotic cells during ontogenesis in order to maintain
cellular homeostasis and to sustain tissue integrity when
cellular and tissue integrity have been compromised. Under-
standing the mechanisms triggered by ionizing radiation can
provide reliable biomarkers important for eff ective triage as
well as tools to modulate tissue reactions after irradiation
to support treatment of radiation casualties (Prasanna et al.
2002). Our eff orts have been recently focused on enterocytes,
which comprise a cellular population with rapid turnover.
Moreover, irradiation of gastrointestinal tract leads to decrease
in enterocyte number with consequent compensatory fl atten-
ing of remaining cells and is observed even after irradiation
by sub-threshold doses. When compensatory mechanisms
become exhausted the gastrointestinal sub-syndrome of acute
radiation sickness/syndrome develops. Th is syndrome con-
stitutes an absolutely lethal clinico-pathological condition,
necrohaemorrhagic enteritis, with unknown causal therapy
(Dri á k et al. 2008).
Ionizing radiation alters activity of mitogen-activated
protein kinases (MAPK), a family of the three distinct kinases
extracellular signal-regulated kinase (ERK), c-jun N-terminal
kinase (JNK), and p38 mitogen-activated protein kinase (p38)
that are activated in response to diff erent extracellular or intra-
cellular stimuli (Johnson and Lapadat 2002, Raman et al. 2007).
In this study, we focused our attention on p38, which is acti-
vated by dual phosphorylation at threonine 180 and tyrosine
182 (Kyriakis and Avruch 2001). Upon activation, p38 translo-
cates into the nucleus, where it phosphorylates transcription
factors and other kinases to adjust cellular functions to stress
stimulation. Th e activation of p38 has been attributed to vari-
ous biological consequences depending on quantitative and
qualitative parameters of stress stimulation and cell or tissue
type. For instance, p38 mediates gamma-irradiation-induced
endothelial cell apoptosis (Kumar et al. 2004) or it is involved
in gamma-radiation-induced G2-phase cell-cycle arrest
in human fi broblast (Wang et al. 2000). Moreover, as
Abstract
Purpose : To examine the p38 mitogen-activated protein kinase
(p38) phosphorylation and transforming growth factor beta 1
(TGF- β 1) expression in rat colon enterocytes after irradiation and
their contribution to pathology of intestinal radiation disease.
Materials and methods : Male Wistar rats were irradiated
with whole body γ -radiation of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10 Gy ( 60 Co, 1.44 Gy.min –1 ). Samples were taken 4 and 24 h
after irradiation, immunohistochemically stained, then p38
phosphorylation and TGF- β 1 expression were measured in
apical and cryptal enterocytes using computer image analysis.
In selected groups, morphometric parameters, mitosis and
apoptosis were evaluated.
Results : P38 phosphorylation integrated optical density (IOD)-
based levels increased 2.4-fold ( p � 0.01) and 3.6 to 22.8-fold
( p � 0.001) in apical enterocytes 4 h after 0.5 Gy and 24 h after 3 –
10 Gy, respectively. TGF- β 1 IOD-based expression increased 3.3-
to 6.9-fold ( p � 0.001) and 1.6- to 4.9-fold ( p � 0.001) in apical
cells 4 h after 0.5 – 2, 4, 5 Gy and 24 h after 6 – 10 Gy, respectively.
No changes were observed in crypts.
Conclusions : We found a chronological and dose-dependent order
of p38 activation and TGF- β 1 expression in apical enterocytes.
Transient up-regulation of p38 and TGF- β 1 signalling observed
4 h after low-dose irradiation may participate in molecular
mechanisms creating cellular over-expression in apical
compartment, while persistent patterns measured 24 h after
high-dose irradiation might provide protection of remaining
cells in order to maintain tissue integrity.
Keywords: p38 , TGF- β 1 , enterocyte , image analysis
Introduction
Direct and indirect damage of cellular components caused
by ionizing radiation induces a wide variety of stress reac-
tions. Th ese response mechanisms have been developed
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p38 and TGF- b 1 in irradiated rat enterocytes 349
demonstrated by in vitro experiments, the activation of p38
upon irradiation seems to be dose-and cell type-dependent
with no, weak, or strong response (Taher et al. 2000, Wang
et al. 2000, Kim et al. 2002, Dent et al. 2003). In those studies
where p38 activation has been observed following exposure
to ionizing radiation, the p38 signalling was dependent on
expression of a functional ataxia telangiectasia mutated
(ATM) protein (Wang et al. 2000, Dent et al. 2003). ATM
accumulates at the site of double-strand breaks, and to date
three links between p38 and ATM/DNA damage have been
proposed (Kharbanda et al. 2000, Choi et al. 2006, Raman
et al. 2007). Th e in vivo situation is more complex. In hetero-
geneous cell populations, such as gastrointestinal tract, the
additional signals activating p38 in enterocytes might come
from adjacent cells, including neighbouring enterocytes,
endothelial cells or fi brocytes. Th ese cells produce cytok-
ines such as transforming growth factor beta 1 (TGF- β 1) in
response to ionizing radiation (Alexakis et al. 2001), which
might regulate superfi cial membrane receptors of the p38
signalling cascade (Howe et al. 2002, Walsh et al. 2008).
Th e aim of this study is to examine the activation of p38
and expression of TGF- β 1 in rat colon enterocytes after irra-
diation and to assess the contribution of these proteins to
early radiation response in vivo.
Materials and methods
Animals Male Wistar rats aged 12 – 16 weeks and weighing 250 – 300
g (Navel, Konarovice, Czech Republic) were kept in an air-
conditioned room (22 � 2 ° C and 50 � 10% relative humidity,
with lights from 7:00 – 19:00 h) and allowed access to stan-
dard food (Velaz, Unetice, Czech Republic) and tap water
ad libitum . Th e rats were divided into 24 groups, each group
consisting of seven animals. Experimental animals were
handled under supervision of the Ethics Committee of the
Faculty of Military Health Sciences (Hradec Kralove, Czech
Republic) and Ethics Committee of the Ministry of Defence
(Prague, Czech Republic).
Irradiation (IR) For IR treatments, the animals were irradiated using a
60 Co unit (Chirana, Prague, Czech Republic) at a dose rate
of 1.44 Gy.min -1 with a target distance of 1 m. Dosimetry
was performed using an ionization chamber (Dosemeter
PTW Unidos 1001, Serial No. 11057, with ionization cham-
ber PTW TM 313, Serial No. 0012; RPD Inc., Albertville,
MN, USA).
Experimental set-up The rats were anaesthetized before irradiation with
a mixture of one volume of Rometar (Spofa, Prague,
Czech Republic) and three volumes of Narkamon
(Zentiva, Prague, Czech Republic). This solution was
injected intramuscularly at 2.8 mg.kg –1 . Under anaes-
thesia, the animals were fastened onto a Plexiglas
underlay (FVZ, Hradec Kralove, Czech Republic) to
assure consistent unilateral front-back exposure and
irradiated by a single dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,
and 10 Gy with two groups sham-irradiated (maximally 2
animals were irradiated each time). Rats were humanely
euthanized by cervical dislocation 4 and 24 h after
irradiation.
Staining Central parts of colon transversum were removed and carefully
fi xed with 10% neutral buff ered formalin (Chemapol, Prague,
Czech Republic). Samples were subsequently embedded
Figure 1. Sample of control rat colon transversum 4 h after sham irradiation. Haematoxylin-eosin stained samples were used to measure length of crypts. Th e length was measured along the cryptal axis (solid arrows). For better resolution, the microphotograph was taken at 200-fold original magnifi cation.
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Figure 2. Sample of rat colon transversum irradiated by 10 Gy 24 h after irradiation. Haematoxylin-eosin stained samples at 800-fold original magnifi cation were used for morphometric analysis of basal lamina length (see also Figure 3). Length of basal lamina was evaluated in 10 apical enterocytes localized at the inner surface (solid arrow).
into paraffi n (Paramix, Holice, Czech Republic), and tissue
sections 5 μ m thick were cut (Microtome model SM2000 R,
Leica, Heidelberg, Germany). Immunohistochemical detec-
tion of TGF- β 1 and p38 phosphorylated at threonine 180
and tyrosine 182 (phospho-p38 Th r-180/Tyr-182) , staining with
haematoxylin and eosin, and immunohistochemical detec-
tion of apoptosis based on labelling of DNA strand breaks
were done.
Immunohistochemical detection of TGF- β 1 and phospho-p38 Thr-180/Tyr-182 Immunohistochemical detection of TGF- β 1 and phospho-
p38 Th r-180/Tyr-182 was performed using a standard peroxidase
technique. After microwave permeabilization (750 W, 2 �
5 min; Electrolux, Hradec Kralove, Czech Republic) and block-
ing the endogenous peroxidase activity for 20 min (1.8 ml of
30% hydrogen peroxide [Vitrum, Prague, Czech Republic] in
Figure 3. Sample of rat colon transversum irradiated by 0.5 Gy 4 h after irradiation. Length of basal lamina was also evaluated in crypts. Th e centre of cryptal base was used as a benchmark (lower dashed line) from which the length was measured (solid arrow). Dashed arrows point out a cluster of apoptotic bodies. A circle indicates a mitotic fi gure.
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p38 and TGF- b 1 in irradiated rat enterocytes 351
Figure 4. Sample of control rat colon transversum 4 h after sham irradiation. To detect apoptosis in apical cells, TUNEL assay detecting DNA fragmentation by labelling the terminal end of nucleic acids was used. TUNEL positivity (brown nuclei) was evaluated at 400-fold original magnifi cation in enterocytes at the inner surface (above dashed line).
100 ml methanol [Kulich, Hradec Kralove, Czech Republic]),
the tissue sections were incubated for 1 h with rabbit poly-
clonal antibody recognizing TGF- β 1 (1:50; Santa Cruz Bio-
technology, Bergheimer, Heidelberg, Germany) and rabbit
monoclonal antibody recognizing phosphorylated p38 (1:50;
Biotech, Prague, Czech Republic) in pH 7.2 phosphate buff -
ered saline (PBS) (Sigma-Aldrich Company, Prague, Czech
Republic) and then washed three times in PBS. All slides were
incubated for 20 min with ready-to-use biotinylated anti-rabbit
antibody (Dako, Prague, Czech Republic). Subsequently, all
slides were incubated with streptavidin horseradish per-
oxidase (Dako) under the same conditions as the secondary
antibody. Excess antibodies and streptavidin horseradish
peroxidase were then washed off with PBS. Finally, 0.05%
3,3 ’ -diaminobenzidinetetrahydrochloride-chromogen
solution (Sigma-Aldrich Company) in PBS containing 0.02%
hydrogen peroxide was added for 10 min to visualize the
antigen-antibody complex in situ .
Figure 5. Sample of control rat colon transversum with negative phospho-p38Th r-180/Tyr-182 expression in crypts 4 h after sham-irradiation. In apical enterocytes, moderate and heterogeneously distributed phospho-p38Th r-180/Tyr-182 positivity was detected. For publication purposes, samples were taken at 200-fold original magnifi cation and counterstained with Harris haematoxylin.
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352 J. Pejchal et al.
Image analysis Stained samples were evaluated using a BX-51 microscope
(Olympus, Prague, Czech Republic) and the ImagePro
5.1 computer image analysis system (Media Cybernet-
ics, Bethesda, MD, USA). Six microscopic fi elds at 400-fold
original magnifi cation were randomly selected from each
rat sample. Image analysis was performed in apical and
cryptal enterocytes in the area of 2100 μ m 2 representing
30 – 40 cells per microscopic fi eld and compartment. Th e
immunoreactive structures were detected within the inverted
Red/Green/Blue scale (RGB) in the ranges: red 75 – 255, green
95 – 255, and blue 100 – 255, where 0 is white and 255 is black.
Subsequently, integrated optical density (IOD) and percent-
age expression (PE) of stained areas within viewing fi elds
were measured. IOD is equal to average optical density �
number of positive pixels (i.e., IOD partially correlates with
PE). Th is parameter refl ects intensity of positivity within a
measured area. Th e scale represents levels from 0 – 2 � 10 5
for a measured area. Within the adjusted range: 0 – 1 � 10 3
corresponds to negativity, 1 � 10 3 to 2 � 10 4 to weak posi-
tivity, 2 � 10 4 to 1 � 10 5 to medium positivity, and 1 � 10 5
to 2 � 10 5 to strong positivity. PE reports the percentage of
positive objects. Th is parameter describes the distribution
of positivity (colour) inside a measured area and refl ects the
distribution of positivity within the epithelium and among
the animals.
Morphometric analysis For morphometric analysis, samples were stained with
haematoxylin-eosin (both Merck, Prague, Czech Republic).
Samples were evaluated using the BX-51 microscope and
ImagePro 5.1 computer image analysis system. Th e length of
25 randomly selected crypts was measured under 100-fold
magnifi cation (Figure 1) and that of 15 randomly selected
Figure 6. Colon transversum of rat irradiated by 0.5 Gy 4 h after irradiation. When compared with control samples, the positivity of phospho-p38Th r-180/Tyr-182 increased in apical compartment while remaining negative in crypts. For publication purposes, samples were taken at 200-fold original magnifi cation and counterstained with Harris haematoxylin.
Table I. Average values for integrated optical density and percentage expression of phospho-p38 Th r180/Tyr182 expression per microscopic fi eld in apical enterocytes � 2 � SE.
4 h after irradiation 24 h after irradiation
Dose (Gy) IOD PE (%) IOD PE (%)
0 17600 � 5000 42.7 � 6.2 500 � 400 1.5 � 1.80.5 41700 � 12900* * 54.0 � 8.7 * * * 600 � 400 1.7 � 1.31 5600 � 3100* * * 17.8 � 5.5 * * * 2700 � 3200 3.5 � 3.92 16500 � 7200 29.5 � 8.2 1100 � 400 4.4 � 2.13 1900 � 1100* * * 5.5 � 2.6* * * 3000 � 1100* * * 10.1 � 3.4* * * 4 2200 � 1000* * * 6.7 � 2.7 * * * 4700 � 2100* * * 16.3 � 5.4* * * 5 4000 � 2500* * * 12.5 � 5.3 * * * 2400 � 1300* * * 6.1 � 3.2* * * 6 19100 � 10100 28.0 � 10.1 2000 � 1200* * * 6.4 � 3.3* * * 7 4700 � 3400* * * 12.4 � 5.6* * * 9700 � 4500* * * 23.4 � 7.2* * * 8 400 � 200* * * 1.5 � 1.2* * * 11400 � 5900* * * 20.9 � 7.5* * * 9 1400 � 800* * * 5.4 � 3.6* * * 7900 � 6300* * * 16.3 � 7.0* * * 10 700 � 300* * * 1.9 � 1.7* * * 1800 � 700* * * 4.8 � 2.3* * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
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p38 and TGF- b 1 in irradiated rat enterocytes 353
closely associated small fragments. Data were expressed as
apoptotic and mitotic index, where apoptotic index � (total
number of apoptotic cell in 50 crypts � 100) / (50 � 28)
and mitotic index � (total number of mitotic cells in 50
crypts � 100) / (50 � 28). Since terminal deoxynucleotidyl
transferase dUTP nick end labelling (TUNEL) can gener-
ate false-positive results (due to DNA fragmentation during
S phase), the method was not used to assess apoptosis in
crypts.
To identify the apoptotic cells among apical entero-
cytes, TUNEL staining was carried out strictly according
to the manufacturer ’ s instructions using in situ cell death
detection kits (Roche, Mannheim, Germany). Briefl y, paraffi n
sections were dewaxed and rehydrated through xylene and an
alcohol series (Kulich). Th e permeability of cell membranes
was increased by incubating the sections in 0.1% Triton X-100
(Sigma-Aldrich Company) with 0.1% sodium citrate (Sigma-Aldrich
basal laminas of 10 apical (Figure 2) and 15 randomly
selected basal laminas of 10 cryptal enterocytes (Figure 3)
was measured under 800-fold magnifi cation.
Apoptosis and mitosis evaluation In crypts, apoptosis and mitosis were measured under 400-
fold magnifi cation using the BX-51 microscope in samples
stained with haematoxylin and eosin. Crypts were selected
for scoring if they represented good longitudinal sections
containing crypt lumen. Total number of apoptotic and
mitotic cells was measured in enterocytes on both sides of 50
longitudinal crypt sections up to the 14th position starting at
the midpoint at the base of the crypt. Th e number of apop-
totic cells was judged subjectively by the size and number
of closely adjacent apoptotic fragments. An apoptotic cell
could be judged as a single large fragment approximately
the size of a neighbouring cell or a cluster of at least three
Table II. Average values for integrated optical density and percentage expression of phospho-p38 Th r180/Tyr182 expression per microscopic fi eld in cryptal enterocytes � 2 � SE.
4 h after irradiation 24 h after irradiation
Dose (Gy) IOD PE (%) IOD PE (%)
0 500 � 100 1.1 � 0.8 200 � 100 0.1 � 0.10.5 700 � 300 2.4 � 1.3 200 � 100 0.2 � 0.21 300 � 100 1.6 � 0.6 300 � 200 0.2 � 0.12 500 � 200 2.4 � 1.3 400 � 200 0.3 � 0.23 300 � 100 1.2 � 0.6 500 � 200 0.3 � 0.14 400 � 100 0.9 � 0.4 400 � 100 0.6 � 0.45 400 � 200 0.9 � 0.3 200 � 100 0.2 � 0.16 400 � 100 2.1 � 1.2 400 � 200 0.5 � 0.37 300 � 100 1.0 � 0.4 300 � 200 0.1 � 0.08 400 � 0 0.9 � 0.3 400 � 200 0.0 � 0.19 400 � 100 1.0 � 0.4 400 � 100 0.1 � 0.010 300 � 100 0.8 � 0.4 300 � 200 0.2 � 0.1
Table III. Average values for integrated optical density and percentage expression of TGF- β 1 expression per microscopic fi eld in apical enterocytes � 2 � SE.
4 h after irradiation 24 h after irradiation
Dose (Gy) IOD PE (%) IOD PE (%)
0 1400 � 300 23.6 � 2.5 1000 � 300 11.7 � 2.90.5 4600 � 1600* * * 29.6 � 5.0 500 � 100* * * 7.8 � 2.0* ** 1 8200 � 3300* ** 34.1 � 5.4* 500 � 100* 7.8 � 2.0* 2 9700 � 3000* ** 37.3 � 5.6* ** 500 � 100* 7.1 � 1.4* 3 1500 � 400 19.6 � 6.0 700 � 200 8.5 � 2.84 2000 � 300* 19.5 � 3.3 900 � 400 8.1 � 2.85 2800 � 700* * * 27.3 � 2.0* 2700 � 1900 17.1 � 4.36 400 � 100* ** 6.4 � 1.8* * * 2000 � 400* * * 31.1 � 2.8* * * 7 400 � 100* * * 4.7 � 1.0* ** 4900 � 1200* * * 38.3 � 3.3* * * 8 700 � 300* * * 10.1 � 2.7* * * 2800 � 700* * * 30.3 � 2.2* ** 9 500 � 100* * * 6.0 � 1.5* * * 1900 � 500* * * 20.9 � 3.4* * * 10 400 � 100* * * 5.9 � 1.4* * * 1600 � 500* * * 20.6 � 2.7* * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
Figure 7. Sample of control rat colon transversum at 200-fold original magnifi cation with week positivity of TGF- β 1 expression in apical enterocytes and negative TGF- β 1 expression in crypts 24 h after sham irradiation. For publication purposes, samples were counterstained with Harris haematoxylin.
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was measured after irradiation by 3, 4, 5, 6, 7, 8, 9, and 10 Gy,
with IOD increasing 5.9-, 9.1-, 4.6-, 3.8-, 18.6-, 22.1-, 15.3-,
and 3.5-fold ( p � 0.001) and PE increasing 6.8-, 11.0-, 4.1-,
4.3-, 15.8-, 14.1-, 11.0-, and 3.2-fold ( p � 0.001), respectively
(Table I).
We observed no signifi cant change of p38 activation in
cryptal compartment (Table II).
Expression of TGF- β 1 In comparison to control values (Figure 7), we measured sig-
nifi cantly increased intensity (IOD) of TGF- β 1 expression in
apical enterocytes 4 h after irradiation by 0.5, 1, 2, 4, and 5 Gy,
increasing 3.3- ( p � 0.001), 5.9- ( p � 0.001), 6.9- ( p � 0.001),
1.4- ( p � 0.01), and 2.0-fold ( p � 0.001), respectively. Dis-
tribution of positivity (PE) was signifi cantly higher 4 h after
irradiation by 1, 2 and 5 Gy, increasing 1.4- ( p � 0.05), 1.6-
( p � 0.001) and 1.2-fold ( p � 0.05), respectively. Irradiation
Company) at 37 ° C for 8 min. After permeabilization, tissue
samples were incubated with 50 μ l of TUNEL reactive mix-
ture at 37 ° C for 60 min in a moist chamber (Bamed, Ceske
Budejovice, Czech Republic) to incorporate fl uorescein
(in TUNEL mixture) into fragmented DNA. Subsequently,
anti-fl uorescein antibody conjugated with horseradish per-
oxidase (from the TUNEL kit) was added for 30 min at 37 ° C
and 3,3 ’ -diaminobenzidinetetrahydrochloride-chromogen
solution was used to visualize DNA fragments. TUNEL-positive
cells were evaluated in 1000 apical enterocytes (Figure 4).
Statistical analysis Th e Mann-Whitney (SigmaStat 3.1, Systat Software Inc.,
Erkhart, Germany) test was used for the statistical analysis
giving mean � 2 � SE (standard error of mean). Th e diff er-
ences were considered signifi cant when p � 0.05.
Results
No animal died prior to scheduled humane euthanasia.
Activation of p38 in rat colon enterocytes 4 and 24 h after irradiation When compared with the control group (Figure 5), signifi -
cantly higher p38 phosphorylation was measured in apical
enterocytes 4 h after the irradiation by 0.5 Gy (Figure 6). IOD
and PE increased 2.4- ( p � 0.01) and 1.3-fold ( p � 0.001),
respectively. On the other hand, decreased activation of p38
was observed 4 h after irradiation by 1, 3, 4, 5, 7, 8, 9, and
10 Gy, with IOD decreasing 3.1-, 9.1-, 8.1-, 4.4-, 3.8-, 49.1-,
12.7-, and 26.9-fold ( p � 0.001) and PE decreasing 2.4-,
7.7-, 6.3-, 3.4-, 3.5-, 28.5-, 7.9-, and 22.7-fold ( p � 0.001),
respectively. Within 24 h after irradiation, higher p38 activation
Figure 8. Under 200-fold original magnifi cation, rat enterocytes showed visible and signifi cant increase of TGF- β 1 expression in apical enterocyte compartment 24 h after irradiation by 8 Gy. In crypts, TGF- β 1 expression remained negative. For publication purposes, samples were counterstained with Harris haematoxylin.
Table IV. Average values for integrated optical density and percentage expression of TGF- β 1 expression per microscopic fi eld in cryptal enterocytes � 2 � SE.
4 h after irradiation 24 h after irradiation
Dose (Gy) IOD PE (%) IOD PE (%)
0 300 � 100 0.8 � 0.4 200 � 100 0.6 � 0.30.5 300 � 200 1.0 � 0.6 200 � 0 0.5 � 0.21 300 � 0 1.2 � 0.6 200 � 100 0.3 � 0.12 400 � 200 1.6 � 1.0 200 � 0 0.6 � 0.33 300 � 0 1.4 � 0.5 200 � 0 0.3 � 0.14 500 � 0 1.1 � 0.5 200 � 100 0.3 � 0.65 300 � 0 1.6 � 0.5 200 � 100 1.0 � 0.46 200 � 100 1.1 � 0.5 200 � 0 0.7 � 0.27 200 � 100 0.5 � 0.1 300 � 0 1.6 � 0.98 200 � 100 0.6 � 0.3 300 � 0 0.3 � 0.59 200 � 100 0.7 � 0.3 200 � 0 0.4 � 0.110 200 � 100 0.4 � 0.2 200 � 0 0.5 � 0.1
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p38 and TGF- b 1 in irradiated rat enterocytes 355
p � 0.01), while decreasing 24 h after irradiation by the same
doses (7.1% and 10.1%, p � 0.05) (Table VII).
Apoptotic index of cryptal enterocytes In crypts, apoptotic index of cryptal enterocytes increased 4
h after irradiation by 0.5, 1, 3, 5, 8, and 10 Gy, being 2.8-, 5.5-,
9.2-, 10.8-, 12.5-, and 14.2-fold ( p � 0.01) higher, respectively,
in comparison with the control value. In the 24-h time inter-
val after irradiation, the index was signifi cantly increased
after irradiation by 3, 5, 8, and 10 Gy, being 1.8- ( p � 0.05),
2.2- ( p � 0.01), 3.0- ( p � 0.01), and 3.4-fold ( p � 0.01) higher,
respectively (Table VIII).
Mitotic index of cryptal enterocytes Mitotic index was signifi cantly decreased 4 h after irradiation
by 3, 5, 8, and 10 Gy and 24 h after irradiation by 5, 8, and 10
Gy, with values decreasing by 75%, 93.8%, 93.8%, and 93.8%
( p � 0.01) and by 76.5%, 94.1%, and 88.2% ( p � 0.01), respec-
tively (Table IX).
Discussion
In this study, we evaluated the eff ect of ionizing radiation
on p38 mitogen-activated protein kinase and on the multi-
potent cytokine TGF- β 1 in rat colon transversum . Both
proteins participate in regulating many vital functions in
vivo. In gastrointestinal tract, TGF- β 1 signalling has been
shown to inhibit intestinal epithelial proliferation and to
modulate diff erentiation of enterocytes (Kurokowa et al.
1987, Barnard et al. 1989, Potten et al. 1995). Th e physiologi-
cal role of p38 is not fully understood, and the kinase plays a
role in diff erent and sometimes even antagonistic responses.
For instance, Guner et al. (2009) demonstrated activation of
p38 in peroxynitrite-induced pro-apoptotic signalling in rat
enterocytes in vitro, while Morris et al. (2010) observed acute
gastrointestinal toxicity in dogs when administered with p38
inhibitors.
According to our results, both activation of p38 and
expression of TGF- β 1 show diff erent profi les in cryptal and
apical compartments after irradiation.
In cryptal compartment, no signifi cant change in TGF- β 1
expression and p38 activation was found 4 and 24 h after
by 6, 7, 8, 9, and 10 Gy decreased TGF- β 1 expression 4 h after
irradiation, with IOD decreasing 3.5-, 3.5-, 2.0-, 2.8-, and
3.5-fold ( p � 0.001), while PE decreased 3.7-, 5.0-, 2.3-, 3.9-,
and 4.0-fold ( p � 0.001). In the 24-hour time interval, TGF- β 1
expression signifi cantly decreased after irradiation by 0.5, 1
and 2 Gy. IOD and PE were 2.0- ( p � 0.001), 2.0- ( p � 0.05),
and 2.0-fold ( p � 0.05) and 1.5- ( p � 0.001), 1.5- ( p � 0.05),
and 1.6-fold ( p � 0.05) lower, respectively, when compared
with control values. In the 24 h time interval, TGF- β 1 expres-
sion was measured signifi cantly higher when irradiated by 6,
7, 8 (Figure 8), 9, and 10 Gy, increasing 2.0-, 4.9-, 2.8-, 1.9-,
and 1.6-fold ( p � 0.001) (IOD) and by 2.7-, 3.3-, 2.6-, 1.8-, and
1.8-fold ( p � 0.001) (PE), respectively (Table III). In crypts,
we measured no change in TGF- β 1 expression either 4 or 24
h after irradiation (Table IV).
Length of crypts In comparison with non-irradiated animals, we observed
decreased length of crypts 24 h after irradiation by 8 and
10 Gy. Cryptal length was reduced by 7.1% ( p � 0.001) and
10.1% ( p � 0.001), respectively (Table V).
Length of basal lamina of 10 enterocytes In apical enterocytes, a signifi cant shortening of basal lamina
was measured 24 h after irradiation by 0.5 and 1 Gy. Th e
length of basal lamina decreased by 10.3% ( p � 0.001) and
7.7% ( p � 0.01). On the other hand, extension of basal lamina
was observed 24 h after irradiation by 3, 5, 8, and 10 Gy. Th e
length of basal lamina increased by 10.3% ( p � 0.001), 7.7%
( p � 0.01), 12.8% ( p � 0.001), and 10.3% ( p � 0.01), respec-
tively (Table VI).
In crypts, extension of basal lamina was measured 24 h
after irradiation by 5, 8, and 10 Gy. Basal lamina extended
by 6.3% ( p � 0.01), 9.4% ( p � 0.001), and 17.2% ( p � 0.001),
respectively, when compared with non-irradiated animals
(Table VI).
TUNEL positivity in apical enterocytes In apical enterocytes, TUNEL positivity increased 4 h after
irradiation by 8 and 10 Gy (16.7% and 21.6%, p � 0.01) and
24 h after irradiation by 0.5 and 1 Gy (15.8% and 10.4%,
Table V. Average crypts length values in rat colon transversum 4 and 24 h after irradiation � 2 � SE.
Length of crypts (μm)
Dose (Gy) 0 0.5 1 3 5 8 10
4 h 227 � 15 221 � 10 216 � 7 222 � 12 236 � 10 214 � 10 225 � 1024 h 216 � 14 232 � 12 218 � 11 205 � 16 207 � 12 184 � 11 * * * 179 � 10 * * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
Table VI. Average basal lamina length values for 10 enterocytes in rat colon transversum 4 and 24 h after irradiation � 2 � SE.
Dose (Gy) 0 0.5 1 3 5 8 10
Length of basal lamina of 10 apical enterocytes ( μ m)4 h 40 � 1 39 � 2 39 � 1 38 � 1 40 � 1 40 � 1 40 � 2
24 h 39 � 1 35 � 1 * * * 36 � 1 * * 43 � 1 * * * 42 � 1 * * 44 � 2 * * * 43 � 2 * * Length of basal lamina of 10 cryptal enterocytes ( μ m)
4 h 66 � 2 64 � 1 65 � 2 65 � 1 67 � 2 66 � 2 66 � 224 h 64 � 2 62 � 2 62 � 2 65 � 2 68 � 2 * * 70 � 2 * * * 75 � 2 * * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
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356 J. Pejchal et al.
the 4 – 24 h time interval and creates cellular over-expression
at lumina surface 24 h after irradiation. Subsequently,
increased number of apoptotic cells and decreased
TGF- β 1 expression measured in apical compartment 24
h after irradiation may function as a compensatory reac-
tion to previous overproduction of diff erentiated cells.
In this context, p38 appears to amplify this eff ect, since
the amount of apoptotic cells measured 24 h after irra-
diation was signifi cantly higher in the 0.5 Gy group than
in the 1 Gy group. Moreover, when using the same in vivo
model, the low-dose p38 activation pattern corresponds
to transient activation/phosphorylation of transcription
factors activating transcription factor-2 (ATF-2), cAMP
response element binding protein (CREB), and myelocy-
tomatosis proto-oncogene (c-Myc) after low-dose irra-
diation (Pejchal et al. 2008). ATF-2, CREB, and c-Myc are
p38 substrates (Noguchi et al. 2000, Kyriakis and Avruch
2001) and regulate genes involved in DNA reparation and
anti-apoptotic activity (Zhou and Walter 1998, Gr ö sch and
Kaina 1999, Amorino et al. 2003, Das et al. 2005, Ma et al.
2007). Th is link indicates that p38 kinase might participate
in molecular mechanisms of adaptive reaction to low-
dose radiation in colon transversum (Wolff 1994, Kadhim
et al. 2004).
Th e biological outcome of the second transient increase of
TGF- β 1 expression observed 4 h after 4 and 5 Gy irradiation
remains elusive. In comparison to 0.5 – 2 Gy irradiation, we
measured no signifi cant cellular or tissue adaptation reac-
tion in apical enterocyte compartment 4 and 24 h after irra-
diation except for extension of basal lamina (see below). On
the other hand, the intensity of TGF- β 1 expression observed
4 h after 4 and 5 Gy irradiation was lower when compared
with the 0.5 – 2 Gy dose interval. Similar results were reported
by Warters et al. (2009), who measured higher TGF- β 1 mRNA
expression in keratinocytes in vitro 4 h after 1 Gy irradiation
than in 0.1 and 5 Gy groups. Th us, in the 4 h time interval, low
doses of ionizing radiation (0.5 – 2 Gy) seem to up-regulate
TGF- β 1 expression more eff ectively and the intensity of its
expression might aff ect cellular/tissue response to ionizing
radiation.
In the high-dose interval (6 – 10 Gy), TUNEL assay indi-
cates that TGF- β 1 might be involved in regulating apoptosis
and may support its protective role in apical enterocytes.
According to our results, irradiation with 8 and 10 Gy was fol-
lowed by decreased TGF- β 1 expression 4 h after irradiation,
irradiation (Figures 5, 6, 7 and 8) despite signifi cant altera-
tion of mitotic, apoptotic, and morphological parameters.
It seems that ionizing radiation does not modulate these
biological processes via either TGF- β 1- or p38-dependent
mechanisms in crypts during the fi rst 24 h after irradiation.
Moreover, similarly to TGF- β 1, phospho-p38 Th r-180/Tyr-182
values were lower in cryptal enterocytes than in apical cells,
thus indicating that the p38 pathway may participate in the
enterocyte diff erentiation process in vivo. Th is is in accor-
dance with in vitro experiments linking p38 to diff erentiation
(Laprise et al. 2002, Grenier et al. 2007, Kuntz et al. 2009).
Th us, ionizing radiation might not increase p38 signalling
and TGF- β 1 expression in crypts in order not to impair the
undiff erentiated status of cryptal cells.
In apical enterocyte compartment, we observed a chrono-
logical and a dose-dependent order of TGF- β 1 expression and
p38 activation after exposure to ionizing radiation. In par-
ticular, the doses of 0.5, 1, 2, 4, and 5 Gy produced increased
TGF- β 1 expression 4 h after irradiation. Th at eff ect seems to
be transient since its values returned to (4 and 5 Gy) or even
dropped below (0.5 – 2 Gy) control values. By contrast, the
dose range of 6 – 10 Gy (Figure 8) increased TGF- β 1 expres-
sion 24 h after irradiation and revealed a rather persistent
expression pattern. Similarly to TGF- β 1, the dose of 0.5 Gy
(Figure 6) led to increased and transient activation of p38 4 h
after irradiation, which was accompanied by a rather persis-
tent activation pattern after high-dose irradiation (3 – 10 Gy).
While the resemblance between TGF- β 1 expression and p38
activation indicates a functional link, the diff erences imply
that TGF- β 1 and p38 function in a more complex signalling
network.
Th e biological outcome of transiently increased p38
activation and TGF- β 1 expression observed 4 h after 0.5
and 0.5 – 2 Gy irradiation, respectively, remains uncertain.
Nevertheless, it is very likely related to protecting apical
enterocytes against apoptosis. Morphometric analysis
shows that up-regulated TGF- β 1 expression in apical
enterocytes 4 h after 0.5 and 1 Gy irradiation is followed
by decreased length of basal lamina of apical enterocytes
24 h after irradiation. Similarly to our model, decreased
width of enterocytes has been observed in rat jejunum 24
h after 1 Gy irradiation (Dri á k et al. 2008). Th us, we may
assume that doses up to 2 Gy stimulate TGF- β 1 production
in apical enterocytes 4 h after irradiation, which seems to
reduce apoptosis in apical compartment at some point in
Table VII. Average TUNEL positivity values for 1000 apical enterocytes in rat colon transversum 4 and 24 h after irradiation � 2 � SE.
TUNEL positivity in 1000 apical enterocytes
Dose (Gy) 0 0.5 1 3 5 8 10
4 h 635 � 38 595 � 44 617 � 43 596 � 72 684 � 70 741 � 16 * * 772 � 22 * * 24 h 673 � 22 779 � 25 * * 743 � 20 * * 679 � 23 660 � 32 625 � 24 * 605 � 42 *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
Table VIII. Apoptotic index of cryptal enterocytes 4 and 24 h after irradiation � 2 � SE.
Apoptotic index (%)
Dose (Gy) 0 0.5 1 3 5 8 10
4 h 1.0 � 0.3 2.8 � 0.4 * * 5.5 � 0.8 * * 9.2 � 0.5 * * 10.8 � 0.4 * * 12.5 � 0.7 * * 14.2 � 1.2 * * 24 h 1.1 � 0.2 1.0 � 0.2 1.5 � 0.3 2.0 � 0.5 * 2.4 � 0.2 * * 3.3 � 0.7 * * 3.7 � 0.6 * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
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p38 and TGF- b 1 in irradiated rat enterocytes 357
transient up-regulations of p38 and TGF- β 1 signalling may
participate in molecular mechanisms of adaptive reaction to
low-dose irradiation creating cellular over-expression in api-
cal compartment of colon transversum . On the other hand,
the persistent patterns observed after high-dose irradiation
might provide protection for remaining cells in order to
maintain tissue integrity.
Acknowledgements
Th is work was supported by the Ministry of Defence of the
Czech Republic through grant MO0FVZ0000501 (institutional
support No. 9079301306023) and grant OVUOFVZ200812 –
RADSPEC. We would like to thank Mrs Š á rka Pr u chov á for
her skilful technical assistance.
Declaration of interest
Th e authors report no confl icts of interest. Th e authors alone
are responsible for the content and writing of the paper.
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Conclusion
Th is study reveals that in vivo there is a chronological and
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crypts. In apical cells, we found transient increase of both
p38 activation and TGF- β 1 expression 4 h after low-dose
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Table IX. Mitotic index of cryptal enterocytes 4 and 24 h after irradiation � 2 � SE.
Mitotic index (%)
Dose (Gy) 0 0.5 1 3 5 8 10
4 h 1.6 � 0.3 1.3 � 0.3 1.2 � 0.2 0.3 � 0.2 * * 0.1 � 0.0 * * 0.0 � 0.1 * * 0.1 � 0.1 * * 24 h 1.3 � 0.3 1.6 � 0.2 1.6 � 0.2 1.2 � 0.4 0.3 � 0.1 * * 0.1 � 0.1 * * 0.1 � 0.1 * *
Signifi cant diff erences among non-irradiated and irradiated animals: * p � 0.05, * * p � 0.01, * * * p � 0.001.
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