Radiatiоn-inducеd aрорtоsis in the small intеstinе оf rats Текст научной статьи по специальности «Биотехнологии в медицине»

Received: 12 May 2017 / Accepted: 2 June 2017 / Published online: 30 June 2017 UDC 616.341-576.31-001.891.53:614.876

RADIATION-INDUCED APOPTOSIS IN THE SMALL INTESTINE OF RATS

Darkhan E. Uzbekov 1, http://orcid.org/0000-0003-4399-460X Kazuko Shichijo 2, http://orcid.org/0000-0003-1370-6865 Nariaki Fujimoto 3, http://orcid.org/0000-0002-8570-4001 Dariya M. Shabdarbaeva 1, http://orcid.org/0000-0001-9463-1935 Nurlan B. Sayakenov \ http://orcid.org/0000-0002-5082-7554 Nailya Zh. Chaizhunusova 4, http://orcid.org/0000-0002-6660-7118 Ynkar O. Kairkhanova 1, http://orcid.org/0000-0001-9533-1723 Aisulu Zh. Saimova 1, http://orcid.org/0000-0002-9564-732X Masaharu Hoshi 3, http://orcid.org/0000-0001-6978-0883 Tolebay K. Rakhypbekov 5, http://orcid.org/0000-0002-5699-3086

1 Semey State Medical University, Department of Pathological anatomy and Forensic medicine, Semey, Kazakhstan;

2 Nagasaki University, Atomic Bomb Disease Institute, Nagasaki, Japan;

3 Hiroshima University, Research Institute for Radiation Biology and Medicine, Hiroshima, Japan;

4 Semey State Medical University, Department of Nutrition and Hygienic disciplines, Semey, Kazakhstan;

5 Semey State Medical University, Semey, Kazakhstan

Abstract

Introduction. According to the literature data, it is known that persons exposed to ionizing radiation, together with a different of damaging effects, particular importance is also attached to the gastrointestinal tract. The dominant role of neutron-activated radionuclide Manganese-56 (56Mn) was noted in the treatises of Japanese scientists who studied the A-bomb effects of Hiroshima and Nagasaki, deserving the interest today.

The research purpose. Investigate the microscopic changes in the small intestine of rats exposed to y- and neutron radiation.

Materials and methods. In experiment, both sexes «Wistar» rats in amount of 36, weighting approximately 220-330 g. Four groups were identified: 1) 56Mn which obtained by neutron activation of 100 mg MnO2 powder using the «Baikal-1» atomic reactor with a neutrons fluence of 4*1014 n/cm2; 2) nonradioactive Mn02; 3) 60Co Y-rays; 4) control group. Necropsy of the animals were on the 3rd, 14th and 60th days after irradiation, then the small intestine removed, after which it was fixed in 10 % formalin. Tissues fragments embedded in paraffin, then sections are manufactured serial transverse 4 )o,m thickness, which were subsequently stained by hematoxylin and eosin (H&E). Specific painting on apoptosis was performed by ApopTag. The difference between samples was examined using the Student's t-test.

Results. Increasing the number of mitotic cells in the small intestine of experimental animals observed on the 3rd day after exposure y- and neutron radiation. Histological analysis of neutron-activated 56Mn showed the high level of apoptosis in the investigated organ. Apoptosis as DNA strand breakage, correlated with cell damage observed on the 14th day after irradiation.

Conclusion. Thus, 56Mn effect on the small intestine of rats showed a high level of risk exposure, which is confirmed by the apoptosis presence.

Keywords: radioactive 56Mn, gastrointestinal syndrome, intestinal crypts, apoptosis.

Резюме

РАДИАЦИОННО-ИНДУЦИРОВАННЫЙ АПОПТОЗ В ТОНКОМ КИШЕЧНИКЕ КРЫС

Дархан Е. Узбеков 1, http://orcid.org/0000-0003-4399-460X Казуко Шичиджо 2, http://orcid.org/0000-0003-1370-6865 Нариаки Фуджимото 3, http://orcid.org/0000-0002-8570-4001 Дария М. Шабдарбаева 1, http://orcid.org/0000-0001-9463-1935 Нурлан Б. Саякенов 1, http://orcid.org/0000-0002-5082-7554 Найля Ж. Чайжунусова 4, http://orcid.org/0000-0002-6660-7118 Ынкар О. Кайрханова 1, http://orcid.org/0000-0001-9533-1723 Айсулу Ж. Саимова 1, http://orcid.org/0000-0002-9564-732X Масахару Хоши з, http://orcid.org/0000-0001-6978-0883 Толебай К. Рахыпбеков 5, http://orcid.org/0000-0002-5699-3086

1 Государственный медицинский университет города Семей,

Кафедра патологической анатомии и судебной медицины, г. Семей, Казахстан;

2 Университет Нагасаки, Институт по изучению заболеваний последствий атомной бомбардировки, г. Нагасаки, Япония;

3 Университет Хиросима, Научно-исследовательский институт радиационной биологии и медицины, г. Хиросима, Япония;

4 Государственный медицинский университет города Семей, Кафедра питания и гигиенических дисциплин, г. Семей, Казахстан;

5 Государственный медицинский университет города Семей, г. Семей, Казахстан

Введение. По данным литературных источников известно, что у лиц, подвергавшихся воздействию ионизирующего излучения наряду с различными повреждающими эффектами особое место отводится и желудочно-кишечному тракту. Доминирующая роль нейтронно-активированного радионуклида - Марганца-56 (56Мп) отмечалась в трудах японских ученых, изучавших последствия атомной бомбардировки в Хиросима и Нагасаки, заслуживающий интерес по сей день.

Цель исследсвания. Изучить микроскопические изменения в тонком кишечнике крыс, подвергавшихся воздействию Y- и нейтронного излучения.

Матеpиалы и методы. В эксперименте использованы крысы обоих полов линии «Вистар» в количестве 36, массой 220-330 гр. Были выделены 4 группы: 1) 56Мп, полученный путём нейтронной активации 100 мг порошка МпО2 на атомном реакторе «Байкал-1» при флюенсе нейтронов 4*1014 н/ом2; 2) нерадиоактивный МпО2; 3) 60Со Y-лучи; 4) контрольная группа. Животных подвергали некропсии через 3, 14 и 60 дней после облучения, затем извлекали тонкий кишечник, после чего фиксировали его в 10 % формалине. Фрагменты тканей заливали в парафин, затем изготовливали поперечные серийные срезы толщиной 4 мкм, которые в дальнейшем окрашивали гематоксилином и эозином (Н&Е). Специфическую покраску на апоптоз осуществляли посредством АрорТад. Разницу между выборками оценивали используя I-критерий Стьюдента.

Результаты. Увеличение количества митотических клеток в тонком кишечнике экспериментальных животных отмечается на 3-е сутки после воздействия Y- и нейтронного излучения. Гистологический анализ нейтронно-активированного 56Мп выявил высокий уровень апоптоза в исследованном органе. Апоптоз как признак разрыва цепи ДНК, коррелирует с повреждением клеток, наблюдаемой на 14-е сутки после облучения.

Вывoды. Таким образом, воздействие 56Мп на тонкий кишечник крыс выявил высокий уровень риска облучения, что подтверждено наличием апоптоза.

Ключевые ^ва: радиоактивный 56Мп, желудочно-кишечный синдром, кишечные крипты, апоптоз.

ТYЙiндеме

РАДИАЦИЯ ЭСЕР1НЕН ЕГЕУК¥ЙРЫКТАРДЫН Ж1Н1ШКЕ 1ШЕГ1НДЕ ТУЫНДАГАН АПОПТОЗ

Дархан Е. Узбеков 1, http://orcid.org/0000-0003-4399-460X Казуко Шичиджо 2, http://orcid.org/0000-0003-1370-6865 Нариаки Фуджимото з, http://orcid.org/0000-0002-8570-4001 Дария М. Шабдарбаева 1, http://orcid.org/0000-0001-9463-1935 Нурлан Б. Саякенов 1, http://orcid.org/0000-0002-5082-7554 Найля Ж. Чайжунусова 4, http://orcid.org/0000-0002-6660-7118 Ынкар О. Кайрханова 1, http://orcid.org/0000-0001-9533-1723 Айсулу Ж. Саимова 1, http://orcid.org/0000-0002-9564-732X Масахару Хоши з, http://orcid.org/0000-0001-6978-0883 Толебай К. Рахыпбеков 5, http://orcid.org/0000-0002-5699-3086

1 Семей каласынын мемлекегпк медицина университетi, Патологиялык анатомия жэне сот медицина кафедрасы, Семей к., Казахстан;

2 Нагасаки университетi, Атом бомбасы эрекетiнен туындаган сыркаттарды зерттеу институты, Нагасаки к., Жапония;

3 Хиросима университет^ Радиациялык биология жэне медицина гылыми-зерттеу институты, Хиросима к., Жапония;

4 Семей каласынын мемлекегпк медицина университетi, Тагамтану жэне гигиеналык пэндер кафедрасы, Семейк., Казакстан;

5 Семей каласынын мемлекеттiк медицина университет^ Семей к., Казакстан

^спе. Эдеби мэлiметтерге сэйкес, иондагыш сэуле эсерiне душар болгандардыц кептеген бYлiндiргiш салдарымен ;оса ас;азанчшек жолдарына да ерекше мэн белшедк Хиросима мен Нагасакидагы атомды; бомбалаудыц салдарын зерттеген жапон галымдарыныц ецбектершдеп нейтронды-белсендi Марганец-56 (56Мп) радионуклидшН басым релi заманауи жагдайда да ^ызыгушылы^ арттырады.

Зеpттеу максаты. Y- мен нейтронды сэуле эсерше ушыраган егеу^уйры^тардыц жНшке шепндеп микроскопиялы; езгерiстердi зерттеу.

Матеpиалдаp мен эдiстеp. Тэжiрибе жYзiнде «Вистар» ту;ымды 220-330 гр салмагы бар аталы; жэне аналы; жынысты 36 егеу^уйры^ пайдаланылган. 4 топ;а iрiктеу жYргiзiлдi: 1) 56Мп, ягни 100 мг МпО2 унтагын «Байкал-1» атом реакторы ар;ылы 4*1014 н/см2 нейтрон флюенсшде нейтронды; белсендiру жYзiнде алынган элемент; 2) бейрадиоактивт МпО2; 3) 60Со Y-сэулелер; 4) бакылау тобы. Жануарларга сэулелеуден кейiн 3-шi, 14-шi жэне 60-шы тэулiктерде некропсия жYргiзу барысында жНшке iшегiн алып, 10 %-ш формалинде фиксациялады;. Тiн фрагменттерiн парафинге куйып, калыццыгы 4 мкм келденец сериялы; кесiндiлер дайындап, эрi карай гематоксилин мен эозинмен (Н&Е) бояды;. Апоптозга арнайы бояуды АрорТад аркылы жYзеге асырды;. Топтар арасындагы сынамаларды СтьюденттН t-елшемi бойынша багалады;.

Нэтижелеp. Тэжiрибелiк жануарлардыц жНшке iшегiндегi митозды; жасушалар саны Y- мен нейтронды; сэулелеуден кейш 60-шы тэулiкте жогарлаганы аны^талган. Нейтронды-белсендi 56Мп-тН гистологиялы; талдауына сай зерттелген агзадагы апоптоздыц жогары децгей тiркелген. Апоптоз ДН^ шбеп бYлiнуiнiц белгiсi ретiнде 14-шi тэултте ацгарылган жасушалар за;ымдануымен байланысты болган.

Ксpытынды. Сонымен, егеукyйрыктардыц жНшке штне 56Мп эсерi апоптоз белсенуiмен расталатын сэулелену ^утнИ жогары децгейш керсетп.

Негiзгi свздеp: радиобелсендi 56Мп, ас;азанчшек синдромы, iшектiк крипталар, апоптоз.

Библиографическая ссылка:

Узбеков Д.Е., Шичиджо К., Фуджимото Н., Шабдарбаева Д.М., Саякенов Н.Б., Чайжунусова Н.Ж., Кайрханова bl.O., Саимова А.Ж., Хоши М., Рахыпбеков Т.К. Радиационно-индуцированный апоптоз в тонком кишечнике крыс / / Наука и Здравоохранение. 2017. №3. С. 32-44.

Uzbelwv D.E., Shield K., Fujiim^ N., Shаbdаrbаеvа D.M., Sayakеnоv N.B., Chaizhunusоva N.Zh., Kairkhanova Y.O., Saimova A.Zh., Hоshi M., Rаkhyрbеkоv T.K. Radiatiоn-inducеd арор^^ in the small intеstinе оf rats. Nauka i Zdravookhranenie [Science & Healthcare]. 2017, 3, pp. 32-44.

Узбеков Д.Е., Шичиджо К., Фуджимото Н., Шабдарбаева Д.М., Саякенов Н.Б., Чайжунусова Н.Ж., Кайрханова bl.O., Саимова А.Ж., Хоши М., Рахыпбеков Т.К. Радиация эсерЫен егеукуйрыктардыц жНшке шепнде туындаган апоптоз / / Гылым жэне Денсаулык сактау. 2017. №3. Б. 32-44.

Introduction. It is known that 56Mn became one of the dominant neutron caused by betairradiator during first few hours following A-bomb explosion in Hiroshima [30]. For the dose-effect relationships in atomic bomb survivors to be applied beyond the radiation quality as a generalized measure of risk assessment at a Gy-equivalent basis of reference radiation, neutrons in atomic bomb radiation in Hiroshima and Nagasaki have been conventionally weighted by a constant value [34]. Therefore, atomic bomb effects on health of survivors have been correlated with delayed Y-rays and neutrons [12]. The accidental high-dose radiation exposure induces a series of injury levels in multiple organs [36]. The highly radiosensitive intestine is an important dose-limitative organ in both total body and abdominopelvic radiation [13]. Most of studies regarding the fast neutron effect have focused at intestinal changes [19].

Nuclear factor is pronounced in gastrointestinal tract those that are exposed to the external environment [20], therefore one of outcomes of radiation effects is gastrointestinal (GI) syndrome [13]. The underlying molecular mechanism of radiation-induced intestinal injury is still not well understood. Some researchers suppose that intestinal stem cells, almost always located in crypts subjected directly to ionizing radiation [18]. It is still unclear whether intestinal stem cell apoptosis or endothelial cell apoptosis is the main factor involved in the initiation and development of radiation-induced GI syndrome. Given that intestinal cell apoptosis has major implications in GI syndrome, radiation oncologists and medical researchers have been seeking radioprotective agents for the intestine that would help to limit intestinal cell death and facilitate intestinal crypt reproduction. Several protective substances that minimize radiation-induced intestinal apoptosis have been known for decades

[11]. Currently, particular interest is a comparative characteristic of microscopic changes in the immune organs of persons exposed to 56Mn and 60Co [5], allowing in the future to work out the diagnostic criteria for assessing of radiation effect factor on the gastrointestinal tract, depending on the cumulative dose.

The objective of study. Our goal has been to identify and compare the microscopic changes in the small intestine of rats after exposure by single 2.0 Gy dose of Y-radiation and neutron-activated 56Mn powder.

Materials and methods. For this study, it was purchased and raised in a the specific-pathogen-free facility six-month-old both sexes «Wistar» rats (Karaganda State Medical University) in an amount of 36 with mean whole body weight 220330 g. All rats were acclimatized for 2 weeks before initiation of experiments and kept under normal conditions and fed pellets concentrated diet and vitamin mixtures. They were maintained at constant temperature (22±1°C) on 8 hour light-dark cycle. Then, rats were allocated into 4 groups. The first group of animals (n=9) were subjected to 56Mn which was obtained by neutron activation of 100 mg of manganese dioxide - MnO2 (Rare Metallic Co., Ltd., Japan) powder using the «Baikal-1» nuclear reactor with neutron flux 4*1014 n/cm2. Activated powder with total activity of 56Mn 2.75* 108 Bq was sprayed pneumatically over rats placed in the special box. The moment of exposition beginning of experimental animals by 56Mn powder is 6 minute after finishing of neutron activation. Duration of exposition of rats to radioactive powder was 3.5-4.0 hours (starting from the moment of spraying of 56Mn powder till surgical extraction of the small intestine) [1].

The second group of rats (n=9) were exposed to not irradiated MnO2. The spray powder was carried out in a chemical box, which contained boxes of 9 rats. Each portion of MnO2 was

sprayed in box with lots of biological objects. Then unirradiated powder and incubated biological objects in a container for hour.

The third group of rats (n=9) were irradiated with a total dose of 2 Gy was performed at a dose rate of 2.6 Gy/min using 60Co Y-ray by czech radiotherapy device «Teragam K-2 unit». Before the exposure, topometry and dosimetry of the rats was performed. After irradiation, rats were taken back to the animal facility and routinely cared. All the experiments were followed our institution's guide for the care and use of laboratory animals. During the exposure, animals were placed in a specially engineered cage made of organic glass with individual compartments for each rat.

The fourth group consisted of control rats (n=9) which were placed on shelves in the same facility and shielded from the radiation. All animal procedures were approved by Ethical Committee of Semey State Medical University, Kazakhstan (Protocol №5 dated 16.04.2014) in accordance with Directive of the European Parliament and the Council on the Office in animals protection. Rats were housed in a moderate security barrier.

The rats were sacrificed on the third, fourteenth, sixtieth day after irradiation and the small intestine was immediately surgically extracted for further histological study. The small intestine sections were deparaffinized and dehydrated in graded 10 % formalin solutions. Paraffin sections performed with 4 |am thickness. For routine pathology, sections were hydrated and stained with hematoxylin-eosin (H&E). Identification of apoptosis was confirmed using a TUNEL technique. The TUNEL assay

(Terminal deoxynucleotide transferase dUTP Nick End Labeling) was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit according to the manufacturer's instructions. The incidence of cell death and number of mitotic cells in the small intestine was quantified by counting the number of cells in each crypt in H&E-stained sections at 40 magnification by light microscopic analysis.

All values were expressed as the mean ± standard error (S.E.) of results obtained from experimental animals per data point. Differences between samples by the level of trait measured quantitatively were estimated for statistical significance using the Student's t-test. A P<0.05 value was considered to be of statistical significance.

Results. In the present study, we performed experiment with neutron-activated 56Mn powder exposed laboratory rats. Although the level of radioactivity received from 56Mn was rather low, the observed biological effects were consistent in experiment. It was previously reported the internal dose estimates in organs of 56Mn-exposed rats. The highest doses were recorded in the small intestine [2]. According to finding, mitosis in this organ was enhanced for an extended period after exposure to 56Mn. For count of mitotic cells in the intestinal crypt was used longitudinal sections of the crypt. On the figure 1, there was a sharp increase the number of mitotic cells in the intestinal crypts of 56Mn-induced (A) and Y-ray-induced (B) rats on the 3rd day after irradiation when compared with MnO2 and control rats.

M.*

Fig. 1. Photomicrograph of rat small intestine. A number of mitotic cells per intestinal crypt were noted in the 56Mn (A) and 60Co (B) groups on the 3rd day after exposure; H&E staining, original magnification x10.

Exposure-related histological changes were the mitotic process could be observed only in rats

noted in the small intestine of rats after neutron exposed to 56Mn (Fig. 2 A, B). and Y-radiation. On the 60th day after irradiation

Fig. 2. Light microscopy of 56Mn-induced rat small intestine on the 60th day after exposure;

H&E staining, original magnification x40.

The small intestine is among the most quickly self-renewing tissues in adult mammals [38]. The number of mitotic cells per crypt in the small intestine are summarized on Table 1. The number

increased in both the 56Mn and 60Co groups on the 3rd day after exposure. While it returned to the control level by 14th day in the 60Co group, it was still high on the 60th day in the 56Mn group.

Table 1.

Number of mitotic cells per crypt in rat small intestine.

№ Group 3rd day 14th day 60th day

1 56Mn 1.81 ± 0.26* 1.14 ± 0.14 2.83 ± 0.24*,#

2 Mn02 1.07 ± 0.20 0.98 ± 0.13 1.71 ± 0.24

3 60Co 2.19 ± 0.25* 0.89 ± 0.11 1.38 ± 0.18

4 Control 0.95 ± 0.18 1.06 ± 0.22 1.32 ± 0.20

Mean ± S.E. * р<0.05 vs. Mn02 and Control, # р<0.05 vs. 60Co

Mitotic index, on the other hand, gradually increased and peaked on the 3rd day after exposure, which coincides with our data showing increases in mitotic cell numbers on the 3rd day in both the 56Mn and 60Co groups. Interestingly, an increase in mitosis was still observed on the 60th day after exposure to 56Mn, while it returned to the control level in the 60Co group, suggesting that the effects of internal radiation of 56Mn were more persistent.

Figure 3 shows that apoptosis was observed in the small intestinal crypts in the rats exposed to neutron-irradiation. On the 14th day after irradiation in rats from the first group, a large number of apoptotic cells was observed in the intestinal crypts, as determined by TUNEL

staining (Fig. 3 A, B). Apoptotic cells different small dimensions comparable with lymphocytes dimensions with high nuclear-cytoplasmic ratio, rounded contours and condensed chromatin and cytoplasm in experimental animals of the first group on the 3rd and 60th day after irradiation, whereas the third group of data changes were identified by three day after irradiation. The distinctive morphological features of apoptosis were used to recognize apoptotic cells. Small clusters of dead cell fragments were assessed as originating from one cell and any doubtful cells were disregarded. Apoptosis was measured on the basis of nuclear image morphology and were able to correlate TUNEL positive staining with measurable nuclear fragmentation.

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Fig. 3. Histologic sections of the small intestine of rats on the 14th day after 56Mn exposure, stained by TUNEL method to make visible the cells containing DNA fragments,

original magnification x40.

A

Apoptotic cells look as the rounded or oval accumulations of intensively eosinophil cytoplasm with dense by the fragments of nuclear chromoplasm.

Table 2 shows the number of mitosis in small intestinal crypt were increased in 2.0 Gy 56Mn

exposed rats on the 3rd and 14th day after internal irradiation and in 2.0 Gy 60Co exposed rat on the 3rd day after external irradiation. Number of apoptosis in small intestinal crypt were increased only in 2.0 Gy 56Mn internal exposed rats on the 14th day after irradiation.

Table 2.

Mitosis and apoptosis in the small intestinal crypt at different days after irradiation.

№

Group

Mitosis

3rd day

14th day

60th day

3rd day

Apoptosis

14th day

60th day

56Mn

increase

increase

increase

Mn02

60Co

increase

Control

1

2

3

4

Discussion

Experimentally confirmed that a certain percentage of Mn enters to organism through absorption in the gastrointestinal tract. If Mn not absorbed in the stomach, it is rapidly absorbed in the small intestine [28]. Microscopic examination which proved that acute radiation intestinal damage triggers apoptosis of intestinal crypt [39], being observed within a period of some hours in rodents [27]. Evidence obtained using genetic modification technology has convincingly shown that intestinal stem cells are columnar cells at the crypt base intermingling with Paneth cells [35]. The molecular determinants of intestinal radiosensitivity and GI syndrome are not well understood. Some believe that damage to stem

cells plays a critical role in this process [32]. Ionizing radiation leads to the exhaustion of the stem cells pool, increases the load on the differentiated cells, resulting in enhanced processes of apoptosis [22].

Previous studies implicated vascular endothelial cell apoptosis in the initiation and development of GI syndrome [10]. The immediate response to damaged DNA is the stimulation of DNA repair machinery and the activation of cell cycle checkpoints, followed by down-stream cellular responses, such as apoptosis. It was observed that 2 Gy irradiation induced apoptosis and cell cycle arrest [9]. Literature data suggest that intestinal crypt stem cell apoptosis dominant over villus vascular endothelial cell apoptosis in

the initiation of radiation-induced GI syndrome [26]. Few studies have focused on a biopolymer whose manipulation significantly regulates GI syndrome via securing stem cell zones and the integrity of intestinal epithelium [23]. Over the past decade, numerous studies have confirmed that multifunctional adaptor proteins have indispensable roles as scaffolds and adaptors in apoptosis-associated signal transduction [24].

Cell death after radiation occurs by mitotic catastrophe and by apoptosis [14]. It should be noted that apoptotic cells are eliminated by the adjacent epithelial cells, endothelial, fibroblasts, macrophages [21, 40]. Apoptosis ensures the removal of dying cells by phagocytosis without inflammation [16]. The most fully the apoptosis role was investigated at tumor growth. Intensification of apoptosis has implications for tumor regression. If the cell is not able to produce apoptosis due the mutation it can start reproducing uncontrollably, resulting to tumors [25]. Radiation-induced apoptosis of intestinal crypts is largely responsible for intestinal tissue damage [29]. In the gastrointestinal system, irradiation induces apoptosis of the small intestinal crypts, contributing to denudation of the intestinal mucosa and reduces the surface for nutrient absorption [31]. The acute morphological changes of intestine by irradiation were consisted of structural changes in the villus-crypt architecture and epithelial transformations associated with radiation-induced apoptosis [13]. Apoptosis is a major pathogenic peculiarity of radiation-induced small intestinal mucosal injury, and apoptosis degree reflects the mucositis degree [7]. Most authors believe that cell death resulting from toxicity of Mn is not a classical apoptosis, and its combination with cessation of ATP synthesis due to mitochondrial damage [33]. Dysfunction or death of intestinal epithelial cells caused by massive apoptosis after radiation influence is considered as dangerous component in the pathogenesis of GI syndrome [15]. The initiation and progression of radiation-induced intestine injury can be caused by disorder of metabolic processes [3, 4, 6, 37] and molecular mechanisms, which form an compounded response [17].

The large increase of apoptotic cells on the 60th day mark in our first experiments revealed a higher turnover of crypt cells for the internal exposure model of crypt cells, as compared to the normal level of apoptosis found in the external

exposure model. As the half-life of 56Mn is three hours, understanding the initial damage to stem cells by internally deposited radioactive materials is crucial.

Although whole-body radiation doses from 56Mn were relatively low, higher internal doses were noted in the small intestine, in addition to significant pathological changes that were more severe and prolonged than the effects of 60Co Y-irradiation. These data may indicate the potential for a high risk of internal exposure to 56Mn, which would have existed in airborne dust after A-bomb explosions in Hiroshima and Nagasaki.

Conclusion. Thus, results shown that number of mitotic cells increased in the small intestine on the 3rd day after 56Mn and 60Co Y-irradiation, but the change persisted only in 56Mn-exposed animals. The histological findings show a significantly higher rate of apoptosis in small intestine for the rats irradiated 56Mn when compared to the other group. Apoptosis is an indication of DNA strand breakage and most likely correlates to the continued cell damage observed beyond 14th day.

Interest conflict

All authors declare no conflict of interest.

Authors contributions:

Uzbekov D. - the practical implementation of all phases of the experiment;

Shichijo K. - the practical implementation of histological staining, acquisition of data;

Fujimoto N. - statistical analysis;

Shabdarbaeva D. - histological analysis and interpretation of data;

Sayakenov N. - the practical implementation of rats necropsy;

Chaizhunusova N. - revision of the manuscript;

Hoshi M. - development of methodology;

Kairkhanova Y., Saimova A.. - collection of literature review;

Rakhypbekov T. - administrative, technical and material support.

The study was conducted according to the scientific project: "The unprecedented multicentering experimental research of ionizing radiation effects on living organisms using a nuclear reactor".

Funding for the project was carried out by Semey State Medical University.

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Correspondence:

Uzbekov Darkhan - PhD student in «Medicine» speciality of Semey State Medical University, Department of Pathological anatomy and Forensic medicine, Semey, Kazakhstan. Address: East Kazakhstan region, 071400, Semey city, Shakarim street, 13 A - 72. Phone: 87222569782, +77055301026 E-mail: darkhan.uzbekov@mail.ru