Article Type: Research Article Article Citation: Cengiz Kurtman, Esra Gümüştepe,
Serdağ Demirören, Fatma Öztürk, Şaban Çakır Gökçe, and Kemal
Özbilgin. (2020). INVESTIGATION OF ZONULA OCCLUDENS-1 PROTEIN LEVEL IN RECTUM
TISSUE AFTER RADIOTHERAPY. International Journal of Research -GRANTHAALAYAH, 8(10),
182-186. https://doi.org/10.29121/granthaalayah.v8.i10.2020.1619 Received Date: 20 September 2020 Accepted Date: 30 October 2020 Keywords: Radiotherapy Acute Side Effect Tight Junction Zonula Occludens Rectum The transmembrane protein zonula occludens of rectal tissue has function to prevents the spread of bacterial toxins into the intestinal mucosa and to systemic circulation. But radiotherapy causes ablation of crypt cell proliferation, mitotic catastrophe, and apoptosis leading to gastrointestinal mucositis. We investigated the acute radiation effect on gastrointestinal mucosa of rectum tissue thickness with immunohistochemistry method for zonula occludens-1 (ZO-1) protein in animal model. A total of 24 healthy Swiss Albino mice were divided into 4 groups, and except control group the groups of 1–3 was exposed to 500 cGy total body irradiation. All rectum tissue samples were taken from the groups of control, 24 h, 72 h, and 168 h after irradiation and stained with hematoxylin and eosin for histochemical examination, and for immunohistochemical staining with anti ZO-1 polyclonal antibody. We observed edema especially in the groups 2 and 3 but not in group 1. Immunohistochemical examination of staining of rectum tissue samples for ZO-1 showed poor staining for control group (1.48 ± 0.06) and group 1 (1.38 ± 0.09) and group 2 (1.50 ± 0.01) but the group 3 (2.12 ± 0.04) samples showed moderate ZO-1 immunostaining. It was found that the amount and thickness of ZO-1 expression increased in the late period for more than 24 hours. The comparison of the values of ZO-1 between the group 3 which is the group in the late period after radiation exposure and control group or group 1 or group 2, showed statistically significant differences (p <0.001). It was concluded that ZO-1 protein may have a role in the side effects of radiation injury, and the understanding of cellular and molecular activity will help us to develop pharmacological modulators to mitigate or treat the injury.
1. INTRODUCTIONThe radiotherapy (RT) is a widely used method in cancer treatment. The main mechanism of action of ionizing rays used in radiotherapy is to stop the proliferation of cells by causing DNA damage [1]. The normal tissues are effacted with the radiation used to destroy cancer treatment [2]. The gastrointestinal tract is one of the radiation-sensitive organs in the body and is characterized by gastrointestinal (GI) complications of radiation in the early stages such as nausea and diarrhea [3]. Endotoxemia and bacteremia may occur in septicemia at a later stage [4], [5], [6], [7], [8]. Intestinal injury occurs in patients receiving abdominal or pelvic radiotherapy and it is characterized by villous atrophy, mucosal edema, ulceration and increased mucosal permeability [9]. Enteral inflammation induced by radiotherapy increases bacterial translocation towards mesenteric lymph nodes [10]. Total body irradiation (TBI) causes ablation of crypt cell proliferation, mitotic catastrophe, and apoptosis leading to gastrointestinal mucositis [11]. TBI causes fatal gastrointestinal injury in high doses such as 14-18 Gy in 7–10 days in mice [12]. In the mouse model, low doses of TBI such as 3–7.5 Gy result in temporary injury to the tight junction between epithelial cells of the intestinal mucosal lining [13]. The function of the epithelial barrier is the first line of defense in the gastrointestinal tract that prevents the spread of bacterial toxins into the intestinal mucosa and into the systemic circulation. The tight junctions (TJ), the highly specialized intercellular junctions, confer epithelial barrier function in the gastrointestinal tract [14]. The TJ, which form most units of apical, define the boundary between apical and basolateral membranes, and are often the speed-limiting factor in the paracellular passage [15]. The TJ has multiprotein complexes made up of both transmembrane proteins such as occludin, tricellulin, different claudins and junctional adhesion molecules (JAMs), as well as peripheral membrane proteins such as zonula occludens (ZO)-1,-2,-3 and cingulin [16], [17]. We investigated the acute radiation effect on gastrointestinal mucosa of rectal tissue with immunohistochemistry method for ZO-1 protein in animal model. 2.
MATERIALS AND METHODS
2.1. ANIMAL
EXPERIMENTS
A total of 24 healthy, 6-8 weeks old, male, adult
Swiss Albino mice, weighing 25-35 g, were obtained from the Ankara University
Experimental Animal Laboratory and used as subjects. The subjects were isolated
from stress and noise and fed with water and food ad libitum at 25°C in a cycle
of 12 h/12 h dark/light conditions before being included in the study. Care of
the animals was performed at the Ankara University Experimental Animal
Laboratory throughout and the study was approved by the Animal Tests Local
Ethics Committee of Ankara University (approval number 2017-21-166, approval
date: 10/18/2017). The mice were divided into 4 groups, each containing 6 mice.
Except the control group, the mice in the experimental Groups 1–3 were exposed
to total body irradiation (TBI) with 6 MV photon using a Varian linear
accelerator device present in the Department of Radiation Oncology of Ankara
University School of Medicine, with a source-to-axis distance of 100 cm, from
anterior (250 cGy) and posterior (250 cGy) fields, and a total dose of 500 cGy
for mid-axis in a single fraction. All mice in the experimental group 1-3 were
injected 45-50 mg/kg intramuscular Ketamine before TBI to provide sedation
during irradiation. The unexposed mice in control group were subjected to
euthanasia after 45-50 mg/kg intramuscular ketamine injection, while the mice
in Groups 1-3 were sedated with 45-50 mg/kg intramuscular ketamine injection 24
h, 72 h, and 168 h after TBI, respectively, and then subjected to euthanasia.
The euthanasia was performed by the method of cardiac perfusion. After
euthanasia, the pelvic region was dissected and the rectum was completely
removed. The tissue samples obtained were embedded into paraffin at 60°C following the
routine light microscopy paraffin tissue method. All rectum tissue
samples were first washed in a solution containing 10% formol and then placed
in screw-cap sampling containers containing 10% formol, with separate boxes
used for every animal. Two sets of serial sections (5-µm thick) were cut and prepared
– the first set was stained with Hematoxylin and Eosin (HE) for histochemical
examination, while the second set of sections were used for immunohistochemical
staining as described below. 2.2. IMMUNOHISTOCHEMISTRY
Formalin-fixed,
paraffin-embedded rectum sections were used for immunohistochemical staining.
Tissue samples were stored at 60°C overnight and then de-waxed with xylene for
30 min. After dehydration of the sections with ethanol, they were washed with
distilled water. Subsequently, the samples were treated with 2% trypsin (ab970,
Abcam, Cambridge, UK) at 37°C for 15 min and incubated in 3% H2O2 solution
for 15 min to inhibit endogenous peroxidase activity. Then, the sections were
incubated with anti-ZO-1 polyclonal antibody (ab216880, Cambridge, UK) in a
1/100 dilution for 18 h at 4°C. They were then given an additional three 5-min
washes in PBS, followed by incubation with biotinylated IgG and administration
of streptavidin peroxidase (Histostain Plus kit Zymed 87-9999, Zymed, San
Francisco, CA, USA). After washing the secondary antibody with PBS three times
for 5 min, the sections were stained with DAB Substrate system containing
diaminobenzidine (DAB, K007, DBS, Pleasanton, CA, USA) to detect the
immunoreactivity, and then stained with Mayer's hematoxylin (72804E, Microm,
Walldorf, Germany) for counterstaining. For positive and negative controls, a
mouse rectum was subjected to the same procedure; however, normal IgG in place
of primary antibody was used as a negative control. All samples were then
covered with a mounting medium (01730 Surgipath, Cambridge, UK) and observed
with light microscopy (Olympus BX-40, Tokyo, Japan). Immunostaining
for ZO-1 expression in the rectum samples were evaluated semi quantitatively
using H-SCORE analysis [18]. The immunostaining intensities were scored as follows: 0
(no staining), 1 (weak, but detectable staining), 2 (moderate staining), and 3
(intense staining). A H-SCORE value was derived for each specimen by
calculating the sum of the percentage of the rectum cells that stained at each
intensity category multiplied by its respective score, using the formula
H-SCORE =
∑Pi (i+1), where i is the intensity score, with a value of 1, 2, or 3
corresponding to weak, moderate or strong staining respectively, and Pi is the
percentage of stained cells for each intensity, varying from 0 to 100%. For
each slide, five different fields were evaluated microscopically at 200×
magnification. H-SCORE evaluation was performed independently by at least two
investigators (KO, SG) blinded to the source of the samples as well as to each
other’s results; the average score obtained by both was considered. 2.3. STATISTICAL ANALYSISAll statistical analyses were performed using IBM, SPSS for
Windows version 20.0 (IBM Corp, Armonk, NY, USA). Kolmogorov-Smirnov tests
were used to test the normality of data distribution. Continuous variables
were expressed as mean ± standard deviation, median (25th-75th percentiles),
and categorical variables were expressed as counts (percentages). Comparisons
of non-normally distributed continuous variables between the groups were
performed using the Kruskal Wallis one-way analysis of variance and
Dunn’s Post Hoc test. A two-sided p value
< 0.05 was considered statistically significant. 3. RESULTSExamination of the
HE stained preparations of rectum samples revealed that; rectum is a hollow tube composed of four
distinctive layers: mucosa, submucosa, muscularis externa, and serosa. The
mucosa of the rectum has, simple columnar epithelium having straight, tubular
intestinal glands with many goblet cells. In the submucosa, we observed light
edema in experimental groups, especially in Groups 2 and 3 but not Group 1
(Figure 1) Immunohistochemical
examination of rectum tissue samples for ZO-1 showed different staining
intensities in the rectum mucosa of four different groups (Figure 2). For each sample, the percentage of rectum
cells stained in each density category was calculated by multiplying with the
corresponding density to obtain an H-SCORE value. The values for each group are
given in Table 1. We observed poor staining for control group (1.48 ± 0.06),
and group 1 (1.38 ± 0.09). The group 2 (1.50 ± 0.01) and group 3 (2.12 ± 0.04)
samples showed moderate ZO-1 immunostaining We noticed that, 24
hours after radiotherapy the staining amount and thickness of ZO-1 decreased
from 1.44 to 1.38 because of the destructive effect of radiotherapy. Then,
because of the repair and protective effect on rectum tissue we noticed
increase to 1.5 at 72 hours and then to 2.12 at 168 hours after radiotherapy,
respectively. The comparison of
the H-SCORE values of ZO-1 positive cells between the control group and the
late exposure to radiation groups was statistically significant (control group
versus group 3; p<0.001). The
expression of ZO-1 in the rectum could not be demonstrated in relation to early
exposure to radiation. On the other hand, it was observed that the amount of
expression increased in the late period for more than 72 hours (Group 3
versus groups 1-2; p < 0.001). No significant difference was
observed between the control group versus 24 hours group 1, and between the control
group versus 72 hours group 2, also between group 1 versus group 2. For ZO-1
positive cells, the main statistically significant difference was seen between
168 hours group 3 versus other groups (p < 0.001). 4. DISCUSSIONThe function of the
epithelial barrier is important because it is the first line of defense in the
GI channel that prevents the spreading of bacterial toxins from the intestinal
mucosa into the systemic circulation [9], [10].
ZO-1 is one of the important peripheral components of the TJ complex [16], [17]. The side effects of
GI secondary to radiotherapy appear in a wide spectrum from mild symptoms to
mortal effects. Total body irradiation (TBI), crypt cell proliferation
ablation, mitotic catastrophe and; GI leads to mucositis [16].
In the mouse model, high doses of TBI (14 - 18 Gy) cause fatal gastrointestinal
damage [17];
low TBI doses (3 - 7.5 Gy) have been observed to cause temporary injuries in
tight junctions (TJ) between intestinal mucosal epithelial cells [11].
In our study; 24 hours after radiotherapy the amount and thickness of ZO-1 decreased
from 1.44 to 1.38 because of the destructive effect of radiotherapy, but
increased to 1.5 at 72 hours and then to 2.12 at 168 hours after radiotherapy
because of the repair and protective effect on rectum tissue. Abdominal RT
applications are known to affect intestinal epithelial cells and cause death,
hypoplasia and ulcerative lesions by pouring cells. The mechanism of GI side
effects observed in the RT result is not fully known. However, it can be
suggested that epithelial barrier disorder may play an important role in the
formation of these side effects. Only one study has been found in the
literature. In this study; In mice treated with ionizing radiotherapy, damage
was observed in ZO-1 at 2 and 24 hours after radiotherapy [18].
In our study, ZO-1 staining measured in the apical region of the rectum
epithelial cells after RT was observed to be the same as the control group
after 24 hours. This result was evaluated as an indication that the effect of
RT on TJ complexes was observed after 24 hours. It was thought that ZO-1
staining increased on 3rd and 7th days after RT application and it could be
associated with proliferation. In the previous study, it has been reported that
the increase in TJ complex's proteins has emerged as a response to barrier
destruction [18]. The ZO-1 and
occludin levels in mouse sperm cells were examined after 100 cGy radiotherapy
that a significant decrease and irregular immunolocalization were observed at 3
and 6 months [19]. In the murine model
assay, the relationship of other proteins constituting the TJ complex with
radiotherapy was increased. On the 4th and 12th day after the exposure to
radiation, the occludin level increased. An increase was observed in JAM-1 on
day 7. Exposure to radiation caused a reduction in the amount of E-cadherin,
but did not significantly affect the amount of fragmented caspase-3 [20].
5. CONCLUSIONIt was observed that RT started to affect ZO-1 expression in the rectum epithelial cells after 24 hours, and the effect was observed on day 3, and its effect continued in 7 days. It was concluded that ZO-1 protein may have a role in the side effects of RT on the intestine. Unraveling the cellular and molecular activity in response to intestinal radiation injury will help understand the mechanisms involved and develop pharmacological modulators to mitigate or treat the injury. SOURCES OF FUNDINGThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. CONFLICT OF INTERESTThe author have declared that no competing interests exist. ACKNOWLEDGMENTNone. REFERENCES
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