Radiation Environment and Medicine 2024 Vol.13, No.1 10–18

Review

Molecular Mechanisms of Acute Radiation Intestinal Injury
and Its Control
Akinori Morita*, Yuichi Nishiyama, Takuma Sakai and Yuichi Higashi
Department of Biomedical Science and Technology, Graduate School of Biomedical Sciences, Tokushima University,
3-18-15 Kuramoto-cho, Tokushima 770-8503, Japann
Received 6 March, 2023; revised 31 August, 2023; accepted 13 October, 2023

The intestinal tract is a representative radiosensitive tissue and a risk organ in radiotherapy that
limits the prescribed dose to tumors in the abdominal and pelvic regions. High-dose radiation
damage to intestinal tissue induces the loss of intestinal epithelial stem cells, and it is now
becoming clear that this process consists of two steps: crypt cell death regulated by p53 andexacerbation caused by inflammatory immune responses. Transcriptional regulators that enhance
p53 function without enhancing apoptosis are effective in controlling the first step. The common
activity of these p53 modulators that promote such functions is that they are able to suppress
apoptosis without impairing p21-mediated cell cycle arrest. Regulating the second step can beachieved by agonists of Toll-like receptor signaling pathways that enhance the priming signal ofpyroptosis and inhibitors that suppress the activity of the inflammasome. In this review, we outline
the molecular mechanisms of each process and discuss strategies for effectively controlling theacute radiation-induced gastrointestinal syndrome.
Key words: radioprotector, p53, p53 modulator, inflammatory response, inflammation control

1. Introduction
Death from exposure to radiation varies with the dose:
death from a whole-body exposure of less than 10 Gy isreferred to as bone marrow death because it is caused by
the hematopoietic tissue damage, whereas death resulting
from exposure to 10 Gy or more results in severe damage
to the intestinal epithelium that precedes the progressionof hematopoietic tissue damage. This process is referred
to as intestinal death1). Radioprotectors (or Radiation
protectors) are drugs that are administered prior to*Akinori Morita: Department of Biomedical Science and Technology, Graduate
School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan
E-mail: morita@tokushima-u.ac.jp
https://doi.org/10.51083/radiatenvironmed.13.1_10
Copyright © 2024 by Hirosaki University. All rights reserved.

exposure to prevent radiation damage and to confer
radioresistance. Radiation mitigators are defined as drugs
that promote tissue recovery after radiation injury, but
in a broader context, they are also sometimes referred toas radioprotectors. Radioprotectors include conventional
radical scavengers that reduce the indirect effects
caused by radiation, and agents that activate biologicaldefense mechanisms have recently been successfully
developed as radioprotectors. It should also be noted
that, regarding the development of radiation mitigators,
several hematopoietic agents have been developed andare generally regarded as promising agents.
T he most impor tant cha llenge in developing
radioprotectors for radiotherapy is to ensure that they
selectively protect normal tissue but not tumor tissue.
The only radioprotector approved for clinical use is the
FDA-approved antioxidant amifostine (also known as WR-

Akinori Morita et al. / Radiation Environment and Medicine 2024 Vol.13, No.1 10–18

2721 or Ethyol), which has been approved for use as aprophylactic agent for dry mouth that selectively protects
salivary glands in the head and neck during radiation
treatments for cancer2, 3). Amifostine is a prodrug in which
the thiol group that is required for radical scavenging iscovered by a phosphate group. After the phosphate groupis removed in the body, it then exerts its radioprotective
effects. If the uptake of amifostine and removal of thephosphate group is slower in head and neck cancers thanin salivary glands, irradiation at a timing within 10 to 30
minutes of administration would selectively protect thesalivary glands. In other words, only when there is a delay
in uptake and dephosphorylation of the drug in the tumor
will it be selective for radiotherapy.
In addition, research on medical countermeasuresagainst acute radiation syndrome (ARS) has been
vigorously pursued in the United States, and since 2015,
the following four hematopoietic agents have been
approved by the FDA; G-CSF (filgrastim), pegfilgrastim (asustained G-CSF product), GM-CSF (sargramostim), andRomiplostim (Nplate; a thrombopoietin receptor agonist)4).
These agents include hematopoietic factors that are
effective in treating chemotherapy-induced bone marrow
suppression in cancer treatment, but when the cancer istreated by radiation alone, it is rare that the hematopoietic
tissue is so extensively irradiated that the treatment
must be interrupted. Therefore, protecting organs at risk
(OAR) near the tumor, rather than hematopoietic tissue,
should be considered in radiation therapy.
2. Protection of the intestinal tract from radiation
by p53 regulation
The intestinal tract is a typical radiosensitive tissue andis the OAR that limits the prescribed dose to tumors in
the abdominal and pelvic region in radiation therapy.
Intestinal tissue damage due to high-dose radiation
leads to the loss of intestinal epithelial stem cells. Highdose radiation exposure causes the radiation-induced
gastrointestinal syndrome (RIGS), which leads tointestinal death 10 to 15 days after exposure throughsymptoms such as diarrhea, dehydration, sepsis, andintestinal bleeding1). In addition, although fractionatedirradiation reduces the extent of normal tissue damage
in radiotherapy, acute and late effects such as intestinal
bleeding, ulcer formation, stenosis, perforation, andintestinal obstruction are known to occur when theintestinal tract is in close proximity to the irradiation
field5, 6). Henson et al. reported that, in a cohort of patientstreated with pelvic radiotherapy, 90% experienced acute
RIGS during treatment, 70% of the patients experienced
either an incomplete resolution of acute RIGS symptoms
or the progression of symptoms originating from acute
RIGS, and by 6 months after treatment, 30% had been

11

Fig. 1. Structure of the intestinal crypt. Intestinal epithelial cells are
replaced by new ones as CBC stem cells actively divide, with the upper
differentiated cells reaching the surface and are then shed from thetips of the villi. The direction of the arrow indicates the direction ofdifferentiation and reversibility. The downward arrows mean that theupper differentiated cells serve as replacement CBC stem cells, when
CBC stem cells are lost and they are able to receive niche factors.

diagnosed with radiation proctopathy5).
Figure 1 shows the structure of the intestinal crypt.
The epithelial surface of the intestinal tissue is composedof crypts and villi, and two types of intestinal epithelial
stem cells are known to exist: crypt base columnar(CBC) cells, which are actively dividing, and +4 cells,
which rarely divide7, 8). CBC cells constitute the crypt
basement by residing between differentiated secretory
cells called paneth cells, and niche factors that are
secreted by these neighboring cells maintain CBC cells
in an undifferentiated state9, 10). Paneth cells express stem
cell growth factors such as EGF, Dll4, and Wnt3, andprovide a niche for CBC cells, primarily by expressing
Wnt10). Since CBC cells are actively dividing while paneth
cells are not, the intestinal epithelial cells that originate
from CBC cells differentiate when they leave the paneth
cells and are no longer able to receive niche factors. CBC
cells actively divide, causing old differentiated cells tomove to the upper crypts and villi with differentiationand subsequently shed from the tips of the villi, replacing
them with new cells within a few days. There are also
secretory progenitor cells, such as +4 and +5 cells,
whose descendants are usually only secretory cells,
but are reserve cells that function as alternative CBC
cells by dedifferentiating, when CBC cells are severely
damaged by radiation or other insults and they are able
to receive niche factors from paneth cells11-14). The cells of
the intestinal crypts are known to contain marker genesthat are highly expressed specifically in each cell: Ascl2,
Lgr5, Olfm4, and Ephb3 in CBC cells; Bmi1, Hopx, Lrig1,

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Akinori Morita et al. / Radiation Environment and Medicine 2024 Vol.13, No.1 10–18

Tert, and Mex3a in +4 cells; Dll1 in +5 cells; and in moredifferentiated cells Alpi and Krt1915-26). By examining theexpression levels of marker genes in samples obtained
from mouse intestinal epithelia that have been damagedby radiation or other agents, it is possible to estimate thepercentage of cells that are severely damaged and thepercentage of cells that are protected from the damage
by administering a protective agent.
The tumor suppressor gene product p53 is involvedin the progression of radiation cell death in bonemarrow and the intestine, but its involvement is quite
different in the two tissues. In the bone marrow, p53
acts to promote radiation-induced apoptosis, while in
the intestinal epithelium it functions as a resistancefactor against nonapoptotic mitotic death. In fact, caudal
half-body irradiation studies indicated that mice thatspecifically lack Trp53 in the intestinal epithelium are
more radiosensitive, whereas Super p53 mice with two
extra copies of Trp53 are radioresistant27). These findings
indicate that there are cell deaths that can be inhibited by
p53 in RIGS.
Therefore, to protect OARs, it is necessary to change
the way p53 is regulated according to its role. We
previously reported that sodium orthovanadate is aneffective p53 inhibitor that suppresses the apoptosispromoting activity of p53 for radiation myelopathy28, 29)
and that 5-chloro-8-quinolinol (5CHQ), a p53 modulator,
enhances the anti-cell death activity of p53 for RIGS
(Fig. 2)30). While the p53 inhibitor sodium orthovanadate
does not show any protective effect against RIGS after
single caudal half-body irradiation31), 5CHQ is one of the
few protective agents that shows efficacy against RIGS
and is a valuable seed compound for promoting thedevelopment of intestinal protective agents.
In mouse intestinal epithelium, 5CHQ enhances theexpression of Cdkn1a, which encodes p21, and suppresses
the expression of Bbc3, which encodes Puma, in a p53dependent manner30). In addition, the expression level of
Lgr5, a marker gene for CBC stem cells, is decreased
by RIGS after caudal half-body irradiation, but 5CHQ
suppresses this effect30). Cdkn1a is known to suppress
nonapoptotic mitotic death in RIGS by arresting the cell
cycle in a p53-dependent manner27), and Bbc3 acts as an
inducer of radiation-induced apoptosis in mouse intestinal
epithelial stem cells32). Research in C57BL/6 backgroundCdkn1a knockout mice, Bbc3 knockout mice, double
knockout mice in both genes, and Trp53 knockout micehas shown that Bbc3 contributes to the promotion of
the radiation-induced cell death of intestinal tissues, as
Bbc3 knockout mice show a prolonged survival after
irradiation, and Cdkn1a contributes to the protection ofintestinal tissues based on the fact that Cdkn1a knockout
mice show reduced survival time33).
Several p53 modulators that confer radioresistance toFig. 2. Two types of p53 regulators that protect radiosensitive
tissues. There are two types of p53 regulators: p53 inhibitors and p53
modulators. p53 inhibitors are effective for treating bone marrow
suppression caused by whole-body exposure, while p53 modulators are
effective for treating intestinal injury caused by local exposure.

cells have been reported in addition to 5CHQ. CHIR99021,
an inhibitor of GSK-3, a serine/threonine kinase, thatspecifically inhibits the acetylation of lysine 120 in the
DNA-binding domain of p53 and PUMA induction by
inhibiting the phosphorylation of the acetyltransferase
Tip60, one of the phosphorylation targets of GSK3. On the other hand, it does not inhibit p21 induction,effectively suppressing the intestinal death caused byabdominal irradiation34). Auranofin, a gold-containing antirheumatic drug, stabilizes p53 by inhibiting proteasomal
degradation through the inhibition of HAUSP7, adeubiquitinase that is involved in regulating the stabilityof p53 35). This agent, like 5CHQ, induces the p53
accumulation and p21 by itself, but not PUMA, and does
not induce the acetylation of lysine 120 in p53, which is
required for PUMA induction, and the resulting reversible
cell cycle arrest by activating the p53-p21 pathway that
inhibits abdominal irradiation-induced intestinal death
and an improved survival36). In addition, antitumor effects
were obtained in mouse abdominal tumor models andAkinori Morita et al. / Radiation Environment and Medicine 2024 Vol.13, No.1 10–18

13

Table 1. Representative p53 regulators and their radioprotective effects
Agents/Compounds
p53 inhibitors

p53 modulators

Characteristics/Mechanisms of action/Effects

References

Sodiumorthovanadate

Inhibition of p53 transcription-dependent and - independent apoptotic pathways,
bone marrow protection, as well as tyrosine phosphatase inhibitor

28, 29Pifithrin αInhibition of DNA-binding and transcriptional activities of p53, bone marrowprotection

42

Pifithrin µ

Inhibition of p53 transcription-independent apoptotic pathway, bone marrowprotection

43

Ex-RAD

Inhibition of p53-dependent apoptosis, bone marrow protection

44, 455-chloro-8quinolinol

Activation of p53/p21 pathway with PUMA suppression, bone marrow andintestinal protection30

CHIR99021

Inhibitor of serine/thereonine kinase GSK-3, p21 induction with PUMA
suppression, intestinal protection

34

Auranofin

Inhibition of p53 proteasomal degradation, activation of p53/p21 pathway without
PUMA induction, intestinal protection

35, 36

RG7112

Activation of p53/p21 pathway through transient disruption of p53-Mdm2
interaction, intestinal protection

38

human malignant colon organoids with the drug alone
and further antitumor effects were observed whencombined with radiation. Pant et al. generated knockin mice in which the p53-Mdm2 negative feedback loop
was disrupted by mutating a p53 response element in theMdm2 P2 promoter37). These mice have normal p53 levels
and activity under normal conditions, are characterized
by increased p53 activity compared to wild-type mice
after stimulation by DNA damage, and are resistant tointestinal death caused by caudal half-body irradiation.
In these mice, they also confirmed that the knockout of
Trp53 only in Lgr5-positive stem cells or the knockout of
Cdkn1a (P21) results in the loss of resistance to intestinal
death 38). In that report, they also reported that thepharmacological transient enhancement of p53 activity inwild-type mice using RG7112, an Mdm2 inhibitor, could
suppress intestinal death38).
One of the issues in radiation protection by p53
regulators is the concern that carcinogenesis is promoted
by suppressing the elimination function of p53 against
oncogenic mutant cells. However, in various mousemodels, it has been shown that the suppression of thethree representative DNA damage responses of p53,
apoptosis, cell cycle arrest, and senescence, does notaffect the p53-mediated inhibition of carcinogenesis,
and that the p53-mediated inhibition of carcinogenesis
is a delayed response that is independent of these threefunctions39-41). These results suggest that the temporary
enhancement of wild-type p53 activity by pharmacologicalmeans may be an effective strategy for alleviating
the problem of intestinal damage in patients who areundergoing radiotherapy in the clinic. Table 1 shows a listof p53 inhibitors and p53 modulators.

3. Protection of the intestinal tract against
radiation through the regulation of immune
mechanisms
Genetic deletion of various inflammation-related genes
in mice has recently revealed the importance of innate
immune responses in RIGS. The molecular structural
patterns that elicit inflammatory responses are classified
into two major groups: pathogen-associated molecularpatterns (PAMPs), which are groups of moleculescharacteristic of exogenous pathogens, and damageassociated molecular patterns (DAMPs), which areendogenous immunogenic molecules that leak fromdamaged cells46). In RIGS, the leakage of self-nucleic acids
as the latter DAMPs is of particular importance.
Concerning pattern recognition receptors (PRRs),
which are activated by DAMPs as ligands, it is important
to consider the intracellular and extracellular distributionof DAMPs and the DAMPs recognition pathway due
to the intracellular location of PRRs. The activation of
intracellular PRRs triggers pyroptosis, an inflammationinduced cell death of macrophages and other cells
responsible for immunity47). Those DAMPs that cannot
be degraded or removed after extracellular leakage aretaken up by surrounding cells by endocytosis and activate
TLR3, TLR7, and TLR9, which are Toll-like receptors(TLRs) in endosomes. The agonists of each are doublestranded RNA (dsRNA) for TLR3, single-stranded RNA
for TLR7, and CpG-DNA for TLR948).
Takemura et al. reported that TLR3-mediated immune
responses are important in the pathogenesis of RIGS49).
Their whole-body irradiation studies of mice lacking Tlr2,
Tlr3, Tlr4, Tlr5, Tlr7, and Tlr9 alone revealed that only
the Tlr3 knockout mice were radioresistant. These mice
showed resistance to RIGS by significantly reducing crypt

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Fig. 3. The role of inflammasomes in pyroptosis. The inflammasome is a platform for the activation of inflammatory
caspases in pyroptosis, a form of lytic programmed cell death. The priming signal pathways and the non-canonicalpathway are also shown.

cell death. They also found that RIGS consists of a twostep pathological process: p53-dependent crypt cell death
resulting in RNA leakage, and p53-independent intestinal
epithelial cell death induced by the leaked dsRNA
through activating the TRIF-RIPK1 pathway, which can
only be utilized by TLR3 in TLR family members. In
addition, an inhibitor of TLR3-dsRNA binding was also
found to improve RIGS by reducing crypt cell death. Of
note, it is known that the TRIF-RIPK1 pathway induces
necroptosis. Indeed, a study using necrostatin-1, a specificinhibitor of RIPK1, had a mitigating effect that improved
mouse survival only when administered post-irradiation(24-72 hours after total-body irradiation), not preirradiation50), which is consistent with the two-step modelof RIGS.
Pyroptosis studies began with the discovery byFriedlander et al. that cell death and the rapid releaseof intracellular contents occurred when primary mouse
macrophages were treated with the lethal anthrax toxin51).
Subsequent studies on the cell death of macrophages
infected with Shigella flexneri led to the discovery again
by Zychlinsky et al. in 199252). That cell death was initiallyconsidered to be apoptotic because of its similar features
to apoptosis, such as caspase-dependence, DNA damage,and nuclear condensation. However, in 2001, DʼSouza et al.
re-defined the term as pyroptosis, derived from theGreek words ʻpyroʼ (fire/heat) and ʻptosisʼ (fall), to denote
caspase-1-dependent inflammatory programmed cell
death53).

The formation of inflammasomes plays an importantrole in pyroptosis (Fig. 3)54). The inflammasome contains a
key adaptor molecule, ASC (Apoptosis-associated Specklike protein containing Caspase recruitment domain),which binds to intracellular PRRs via its Pyrin domain,
and at the same time, ASC binds to procaspase-1 via its
own Caspase recruitment domain. The mechanism bywhich inflammatory cytokines such as IL-1β and IL-18
are secreted extracellularly has long remained unclear
because they lack secretory signaling sequences, but thediscovery of Gasdermin D (GSDMD), a plasma membranepore forming factor that is cleaved and activated by
caspase-155) explains this dilemma regarding pore
formation during pyroptosis is now considered to be themajor secretory mechanism of inflammatory cytokines.
After the discovery of GSDMD, a new definition ofpyroptosis as cell death via pore formation by a group of
GSDM molecules was proposed56).
In RIGS, the AIM2 inflammasome, which is formed via
the PRR, AIM2 (Absent in melanoma 2), that recognizes
double-stranded DNA (dsDNA), is known to regulate cell
death and tissue damage in the intestinal epithelium57).
In that study, the knockout of either caspase-1, AIM2, orthe adaptor molecule ASC, a molecule within the same
inflammasome platform, suppressed radiation-induced
intestinal death, suggesting a close relationship between
RIGS and inflammatory cell death. Furthermore, thestudy also showed that intestinal resistance is maintainedin mice in which caspase-1 is specifically lacking in theAkinori Morita et al. / Radiation Environment and Medicine 2024 Vol.13, No.1 10–18

intestinal epithelium, indicating that inflammasomemediated cell death occurs in the intestinal epitheliumin RIGS. It has also been reported that NLRP3 (NACHT,
LRR and PYD domains-containing protein 3) knockout
mice have also been reported to show resistance in a 9.5
Gy whole-body irradiation test58).
The mechanisms responsible for pyroptosis havebeen investigated in detail in the case of TLR signaling
pathways. The process is known to occur via a twostep process in the canonical pathway 47, 54). The firststep of the process requires “priming” by the bindingof TLRs to their ligands, which activates NF-κB andinduces the expression of several proteins that form theinflammasome complex and precursors of inflammatory
cytokines such as pro-IL-1β and pro-IL-18. In the second
step of the process, caspase-1 is activated and GSDMD,
pro-IL-1β, and pro-IL-18 are cleaved, leading to pyroptosis
with cell lysis. The pathway in which intracellular PAMPs
directly activate caspase-4/5 without the formation of
inflammasomes and lead to pyroptosis is referred to as
the non-canonical pathway. The molecular mechanism
responsible for the transition from the first to thesecond step in RIGS and the nature of the target cells
are currently poorly understood, but since the knockoutof intracellular PRRs such as AIM2 and NLRP3 resultsin radioresistance, the second step in pyroptosis can beconsidered to be an exacerbating factor in RIGS.
On the other hand, the first-step priming signals are
considered to be signals that contribute to intestinal
epithelial cell resistance in RIGS, as various agoniststhat enhance the pathways confer radioresistance.
The full activation of the canonical pathway requires
transactivation by NF-κB and others, and this action also
appears to cause the activation of factors that confer
radioresistance. To date, agonists of TLR2/659-63), TLR464-68),
TLR569-71), and TLR972) have been reported to exhibit
protective or mitigating effects against RIGS.
In addition, if the extent of RIGS-induced damage issevere, the intracellular PRRs, AIM2 and NLRP3, may
be activated without proceeding through the first steppriming process, thus activating signals that exacerbate
the inflammatory response. Under these circumstances,
caspase-1, which is responsible for inflammasome
activity, may be a potential drug target for therapies
that antagonize RIGS. Several inflammatory caspase
inhibitors have been designed as potential therapeutics
for the treatment of inflammatory diseases. Only a
few have been clinically tested due to barriers such as
inadequate efficacy, poor target specificity, or severe side
effects, resulting in the fact that no synthetic caspase
inhibitors have yet been approved73). Synthetic caspase
inhibitors are classified as peptide-based inhibitors,
peptidomimetic inhibitors, and nonpeptidic inhibitors.
Inhibitors that target caspase-1, such as Ac-YVAD-

15

CMK74) and Z-YVAD-FMK75) for peptide-based inhibitors
and VX-765 (Belnacasan)76), VRT-043198 77), and VX740 (Pralnacasan)78) for peptidomimetic inhibitors havebeen actively studied. For the case of RIGS, VX-765 has
actually been reported to suppress intestinal death caused
by abdominal irradiation79, 80). In addition, although thefollowing might be considered as indirect effects, theefficacy of Micheliolide81), Rosiglitazone82), Resveratrol83),
which regulate NLRP3, or 5-androstenediol84), which
regulates AIM2, against RIGS has been reported.
4. Summary
Figure 4 summarizes the molecular mechanism for RIGS
and its regulators. The molecular mechanism responsible
for radiation acute intestinal injury is outlined as a twostep process that begins with a p53-regulated process,followed by an inflammatory exacerbating process. We
also introduced the concept that the enhancement of p53
function that does not enhance apoptosis can be effective
in counteracting the first step, and that regulating
the priming signal of pyroptosis and the inhibition of
inflammasomes activity are effective in counteracting
the second step. The suppression of radiation-induced
intestinal damage by p53 modulators has shownpromising results regarding the selective protection of
normal tissues in a mouse model. The issue of whether
normal tissue selectivity can be achieved by regulating
the second step of the process awaits further research.
We hope that further studies on the pathologicalmechanisms of bot h processes will lead to t hedevelopment of more effective methods for controllingradiation intestinal injury.
Conflicts of Interest
The authors declare no conflict of interest.
Funding
Some of the research described in this review was
supported in part by JSPS KAKENHI Grant Number
24689050, 16K10396, 19K17143, 19H03604, 21K07644, and
23H02859, and by the Program of the Network-type Joint
Usage/Research Center for Radiation Disaster Medical
Science.
Abbreviations
ARS, acute radiation syndrome; OAR, organs at risk;
RIGS, radiation-induced gastrointestinal syndrome;
CBC, crypt base columnar; 5CHQ, 5-chloro-8-quinolinol;
PAMPs, pathogen-associated molecular patterns; DAMPs,
damage-associated molecular patterns; PRRs, pattern

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Fig. 4. The role of inflammasomes in pyroptosis. Details of p53-inhibitable cell death(s) are unknown, but theoverexpression of p53 results in resistance to RIGS27). The role of caspase-1 is not limited to macrophages; the intestinal
epithelial-specific deficiency of Casp1 also confers resistance to RIGS57). This death is herein referred to as caspase-1dependent cell death. A plausible molecule that could act in both the first and second steps could be considered AIM2.
In that case, the endogenous dsDNA would act in the first step and the exogenous dsDNA in the second step.

recognition receptors; TLRs, Toll-like receptors; dsRNA,
double-stranded RNA; ASC, apoptosis-associated specklike protein containing caspase recruitment domain;
GSDMD, Gasdermin D; AIM2, Absent in melanoma 2;
dsDNA, double-stranded DNA; NLRP3, NACHT, LRR and
PYD domains-containing protein 3.
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