Activation of p38 MAPK and expression of TGF-β1 in rat colon enterocytes after whole body...

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: [email protected]

( 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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350 J. Pejchal et al.

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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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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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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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.



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.



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* * *


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.



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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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 * * *


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 * * *


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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 *


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 * *


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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

dose-dependent order of p38 activation and TGF- β 1 expres-

sion in apical enterocytes even as no change is observed in

crypts. In apical cells, we found transient increase of both

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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 * *


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Activation of p38 MAPK and expression of TGF-β1 in rat colon enterocytes after whole body...

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