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Abstract

Radiation damage assessment of the small intestine is important in nuclear accidents or routine radiotherapy of abdominal tumors. This article reviews the clinical symptoms and molecular mechanisms of radiation-induced small intestinal damage and summarizes recent research on biomarkers of such damage. Citrulline is the most promising biomarker for the evaluation of radiation-induced small intestinal damage caused by radiotherapy and nuclear accidents. This article also summarizes the factors influencing plasma citrulline measurement investigated in the latest research, as well as new findings on the concentration of citrulline in saliva and urine after different types of radiation.

Keywords

biomarker small intestine damage citrulline radiation

Introduction

Nuclear weapons used in wars, accidents at industrial and nuclear power plants, accidental exposures from medical sources of radiation, environmental factors, and nuclear terrorist attacks are some of the ways humans face radiation-related injuries. Where a radiation source is known, every effort should be made to avoid exposure to radiation and shorten the exposure time, avoid the radiation source, and seek shelter. Once the human body is exposed to radiation, appropriate screening and medical aid should be applied immediately. In the various scenarios mentioned above, because of differences in factors such as gender, age, radiation dose and rate, radiation type, external or internal exposure, health background, lifestyle habits, etc. identifying the irradiated population and applying timely medical assistance are very complex tasks. A radiation biomarker (a biomarker) refers to a class of substances that can be utilized to indicating the interaction between biological systems and radiation. The ideal radiation biomarker can reflect the radiation dose received by the individual, type of radiation, exposure time, damaged organ, and pathological stage of damage. Radiation biomarkers can be used to screen individuals exposed to radiation during a nuclear accident, assess health risks, and provide timely mitigation or preventive measures.

In recent decades, research on radiation markers has become increasingly detailed. Candidate genes for various radiation markers screened from human blood have been systematically reviewed by Lacombe.¹ However, because the sampling and research methods of many studies are different, further research is required. The gastrointestinal (GI) tract consists of radiation-sensitive organs and it is the first to have a systemic crisis after being exposed to radiation, which affects nutrition intake and even causes systemic inflammation. Gut health assessment should be given high attention in the early stages of assisting irradiated persons. In addition, in the radiotherapy of celiac tumors, the intestinal toxicity caused by irradiation is the biggest cause of normal tissue damage and limit radiation efficiency dose. Therefore, it is necessary to pay attention to the evaluation of intestinal radiation damage in daily radiotherapy practice. This article reviews the advances in the discovery and evaluation of biomarkers of small intestine damage caused by ionizing irradiation and provides a novel review of plasma citrulline research that is most promising for clinical application.

Radiation Damage of the Small Intestine

The intestines are the foci of radiation protection. The GI tract is a normal entrance and absorption pathway for nutrients, has a high degree of structural complexity, and constituted from multiple cell types, each performing a different function. It is fundamentally sensitive to a variety of pathogenic microorganisms, chemical, and radiation attacks. Studies have found that there are many factors that affect the initial radiation dose of small bowel injury. First, different types of radiation cause different degrees of intestinal damage. For example, neutron radiation causes more serious intestinal damage than gamma photon radiation.² Secondly, the volume of the small intestine irradiated is also an important factor, the larger the exposure volume, the more serious the damage is.^3
-5 Irradiated volume of the small intestine affects not only acute small intestine injury but also chronic damage. Emami et al⁶ evaluated the radiation dose associated with delayed toxicity of the small intestine. When one third of the small intestine is irradiated, the TD5/5 (5% chance of injury showing up over the next 5 years) and TD50/5 (50% chance of injury showing up over the next 5 years) were estimated to be 50 Gy and 60 Gy, respectively. While the TD5/5 and TD50/5 of whole organ irradiation were 40 Gy and 55 Gy. In addition, the fixed parts of the small intestine (such as the duodenum and the terminal ileum) are more sensitive to radiation.⁷ Because the growth of small intestinal epithelial cells has a circadian rhythm, the time period of exposure during the day can also affect the severity of the injury.⁸ These uncertainties make it impossible to have a clear threshold for radiation damage to the small intestine. Acute radiation damage occurs when the systemic dose exceeds 2 Gy, and as the level of radiation exposure increases, the severity of symptoms also increases.⁹ Studies have also found that doses as low as 1.5 Gy can cause the prodromal stage of nausea, vomiting, and gastric cancer.² In some experiments the dose that caused the collapse of the GI system was 6-10 Gy.¹⁰ While some researchers believe that the losing of intestinal crypt cells and breakdown of the mucosal barrier occurs between 5-12Gy.¹¹ Moreover, from the experimental data of the monkeys listed in Table 1, it can be seen that if different observation time points are selected, different conclusions will be obtained.

Table 1.

The Influence of Time Course and Dose on the Observation of the Severity of Small Bowel Injury Caused by Radiation in Non-Human Primates.

Species (radiation condition)	Dose	Time course	symptom
Rhesus macaque¹²(X-ray; Total body irradiation)	10.0 ∼14.0 Gy	Day 4 post-TBI on average, peaking at day 7	Diarrhea
	10.0Gy	Day 5 on average, peaking at day 7	Grade 2 Dehydration
	14.0 Gy	Peaking at day 7	Grade 3Dehydration
	10.0 ∼14.0 Gy	Day 7 on average	Crypt numbers reaching 10% loss of pre-irradiation body weight.
	10.0Gy	On day 7 post-TBI	Crypt numbers decreased to 30% of control.
	10.0Gy	On day 10 post-TBI	Crypt numbers decreased to 24.8% of control.
	10.0Gy	On day 15 post-TBI	Crypt numbers restored to 44% of control.
	11.5Gy	On day 7 post-TBI	Crypt numbers decreased to 6% of control.
	11.5Gy	On day 10 post-TBI	Crypt numbers restored to 28.8% of control.
	11.5Gy	On day 15 post-TBI	Crypt numbers restored to 25% of control.
Rhesus monkeys¹³ (X-ray; Total abdominal irradiation)	9.5Gy	Appeared initially at 5 days, continued through 14 days	Diarrhea
	9.5Gy	At 7 and 14 days after post TAI, but not at 35 days after TAI	Crypts/millimeter were reduced significantly relative to baseline controls.

The pathophysiological mechanism of gastric syndrome caused by radiation is complex, involving the loss of crypt cells, reduction in the number of intestinal villi, poor regeneration of intestinal stem cells after irradiation, and systemic inflammatory response syndrome (SIRS) caused by a variety of cytokines and growth factors.^14,15 In addition to the intestinal damage caused by the direct effect of radiation, it is generally believed that the bystander effect caused by radiation will also cause intestinal damage¹⁶ through 2 pathways of intercellular gap and paracrine.¹⁷ Some irradiated cells can cause damage to neighboring cells through soluble components^18,19 or release of exosomes. In 2007, Gaugler’s research demonstrated that in high-dose radiation in vitro experiments, irradiated EC epithelial cells plays essential role in the initiation of the pathogenesis of intestinal damage to radiation, ie, epithelial cell lethality. A similar phenomenon was also discovered in the study when using Dark Agouti rats for fractional exposure experiments.²⁰ In recent years, the rise of organoids has led to new methods for the study of bystander effects. Enteroids, small intestinal crypt organoids, consist of a 3D epithelial monolayer that maintains crypt-villus architecture with replicating ISC intestinal stem cells that differentiated into the major small intestinal epithelial lineages.^21,22 Using Enteroids, Leonetti’s research²³ have found Ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. The rapid turnover of intestinal epithelial cells results in the intestinal mucosa being particularly sensitive to high radiation exposure during radiation therapy or any other nuclear exposure. Therefore, maintaining intestinal homeostasis is essential in order to resist radiation-induced GI damage.²⁴ After being exposed to radiation, living organisms often show active or passive changes in biological macromolecules such as nucleic acids, proteins, and metabolites in cells, organs, and body fluids. We wish those biological macromolecules can reflect the exposure time and radiation characteristics (type, dose, dose rate, etc.), and changes in biological macromolecules in such as injured organs can provide urgently required information to emergency medical service professionals.

Clinical Symptoms of Small Intestinal Damage Caused by Radiation

The small intestinal epithelial cells are constantly renewed, and the cells migrate from the intestinal crypt along the sides of the villi and eventually fall off at the top. Controlling cell adhesion during cell migration, division, and differentiation is essential to maintain its healthy and sustainable regeneration.²⁵ Complex gene expression networks^26,27 control the steady-state of multicellular proliferation, starting from stem cells.²⁸ These overly complex control networks are most vulnerable to radiation damage.²⁹

Radiotherapy of abdominal and pelvic malignancies usually causes severe intestinal toxicity,³⁰ which is an important clinical problem that restricts the dose determination in radiotherapy. The total risk of this complication depends on the stage of cancer, patient age, GI condition, and radiation type, dose and fractionation.³¹ Radiation destroys and depletes stem and immature cells, making it impossible for the body to fully compensate for defects caused by the exfoliation of differentiated cells. This results in a change in the morphology of the mucous membrane in an inflamed form. In turn, the rapid natural renewal of the intestinal mucosa makes these cells particularly vulnerable to cytotoxicity treatment.

Mucositis, also known as mucosal barrier damage, has complex pathological and clinical manifestations.³² It is characterized by physiological changes in the epithelial layer—from erythema to ulcers. Mucositis is also one of the most debilitating side effects of radiotherapy and chemotherapy.³³ The epithelial barrier lining the GI tract is composed of a single layer of epithelial cells,³⁴ forming a mechanical barrier that separates the inside of the human body from the outside world. Mucosal damage disrupts the body’s natural barrier against infection. In addition, a weakened immune system is a factor leading to the dynamic development of infection. Inflammation, the loss of mucosal integrity, and neutropenia increase the risk of local bacterial, fungal, and viral infections.

Molecular Mechanism of Small Intestine Damage Caused by Radiation

Owing to the particularity of the small intestine, there is currently no molecular model for small intestinal mucosal damage caused by radiation, but it is generally believed that the damage process of oral epithelial cells should be consistent with that of small intestinal mucosal epithelial cells.³⁵ Treister and Sonis³⁶ observed that the general cellular process of mucosal injury involves not only the damage to epithelial cells, but also the participation of other molecular processes. Recent studies have shown that the mechanisms involved in the pathogenesis of mucositis are more complex than direct damage to the epithelium alone. Radiation therapy with multiple doses of radiation will trigger a series of biological events in the intestinal villi epithelial cells. It is generally believed that the mucosal damage caused by radiation can be divided into 5 stages, according to the model introduced by Sonis.^37,38 Different regions of the mucosa may undergo each stage of damage independently.^37,39

a. Initial stage: Radiation directly damages epithelial cells, the basement membrane, and submucosal blood vessels. The direct effects of radiation cause DNA damage and the death of epithelial cells. The reactive oxygen species generated by the indirect effects of radiation are also considered to play an important role in the occurrence of mucosal damage.⁴⁰ The formation of these lesions leads to the activation of nuclear factor κB (NF-κB).⁴¹

b. Inflammatory factor stage: This stage involves the activation of inflammatory cytokines such as interleukin 1, tumor necrosis factor alpha (TNF-α), and interferons (IFN), and the initiation of angiogenesis. During epithelial cell injury and death, the second messenger is activated, leading to the upregulation of pro-inflammatory cytokines and tissue damage.⁴² The activation of messenger molecules causes the onset of inflammation. Intestinal changes at this stage include intestinal epithelial cell apoptosis and morphological changes of the small intestinal villi.

c. Signaling and amplification stage: This stage involves the enhanced release of cytokines, leading to mucosal damage and loss of its integrity and continuity. Primarily, macrophages begin to produce pro-inflammatory cytokines such as TNF-α, and activate molecular pathways that amplify mucosal damage. The cascading effect of inflammatory factors leads to the increased involvement of immune cells and apoptosis of mucosal epithelial cells.

d. Barrier dysfunction stage: At this stage, the small intestine produces mucosal ulcers. The ulcer phase is characterized by the disruption of the continuity of the epithelial barrier. The disruption of barrier function is the result of the combined action of epithelial cell apoptosis, the development of mucosal ulcers, inflammatory cell infiltration, dysfunction of the local immune response mechanism, and microbial translocation (viz., of bacteria, viruses, and fungi). Metabolites of intestinal microorganisms are also one of the causes of inflammatory cell infiltration.

e. Recovery stage: Owing to the continuous differentiation and proliferation of mucosal cells, the integrity and continuity of the epithelium and the normal functioning of the small intestine villi are restored.⁴³ At this stage, the microvessels in the villi of the small intestine are also recovered further.⁴⁴

Biomarkers of Radiation Damage in the Small Intestine

In recent decades, biomarkers of radiation damage in the small intestine have been researched extensively, and many methods and candidate biomarkers have emerged. Studies on the small intestine generally focus on the unique features of the small intestine, such as absorption, barriers, and amino acid synthesis. Owing to technological progress, various advanced methods such as mass spectrometry and nucleic acid sequencing have been incorporated in the research methods in recent years. The use of various omics methods has promoted the birth of more noninvasive methods. However, there is currently no small intestinal radiation damage biomarker approved by competent authorities, and there is not even a “gold standard” in the industry. Additionally, among the several possible candidate biomarkers currently under investigation, almost all of them fail to meet the screening requirements of ideal radiation biomarkers specific for radiation types. Only the radiation dose has a good correlation within some candidate biomarkers. Many potential radiation biomarkers targeting the small intestine have been proposed, such as diamine oxidase (DAO) calprotectin and gut flora. DAO^45

-48 is a highly active intracellular enzyme in the upper villi of the small intestine of humans and mammals. It is closely related to the integrity and damage of the intestinal mechanical barrier. However, the low level of DAO in the blood makes it difficult to detect, and it is easily confused with heparin in the blood.^49,50 Calprotectin^51,52 is a calcium- and zinc-binding protein with a molecular weight of 36 kD. The concentration of calprotectin in feces has been identified as a sensitive biomarker for intestinal inflammation. It is highly sensitive and noninvasive, but low in specificity and cannot distinguish the anatomical site of intestinal injury.^53,54 The composition of the microbiome in gastrointestinal tract is unique to an individual. However, it is not fixed and can be altered according to various factors such as changes in environment, drugs, and diseases. Studies have shown that radiation can cause significant changes in the gut microbiota.^55,56 And microbiome plays an important role in the pathogenesis of radiation-induced intestinal damage.⁵⁷ Although compared with the sham irradiated control, the intestinal microbiome of radiated one shows a reduction of specific flora,^58,59 the amount of microbe reduction cannot linearly indicate the radiation dose. Moreover, certain pre-existing pathology can also affect the specificity of intestinal flora as a marker of radiation damage.⁶⁰ The data form patients that scheduled to receive abdominal radiotherapy in 3 different clinical trials,^61
-63 and normal C57 mice in an abdominal irradiation experiment⁶⁰ shown that the microbiota profile changed greatly before and after irradiation. However, there were discrepancies regarding the nature of these alterations between studies.⁶⁴

Citrulline as Biomarkers of Radiation Damage in the Small Intestine

Features of citrulline secretion in the small intestine

Citrulline is currently the most in-depth researched candidate that meets most requirements (tissue specificity, volume-response relationship, etc.). The citrulline test mainly evaluates the loss of intestinal epithelial cells, which is an important manifestation of acute and chronic intestinal radiation damage. There are currently 2 pathways found in the synthesis of citrulline in vivo, and these 2 pathways are mainly completed in the small intestine. The first is the synthesis of citrulline from glutamine, which requires 5 mitochondrial enzymes: phosphate-dependent glutaminase (PDG), pyrroline-5-carboxylic acid synthase (P5CS), ornithine aminotransferase (OAT), Ornithine carbamyltransferase (OCT), and Carbamoyl phosphate synthase I (CPSI), of which P5CS is the key regulator^65

-68 and unique to small intestinal epithelial cells.^66,69,70 Proline synthesis of citrulline and arginine is another important pathway for citrulline synthesis. This pathway involves 4 mitochondrial enzymes,⁷¹ namely proline oxidase (PROox), OAT, OCT, and CPSI. Although the key regulatory enzymes of this pathway, PROox and CPSI,^65,72 are also found in the liver and kidneys, the activity of PROox in the small intestine is relatively high, that is, 10 times and 6 times higher than that in liver and kidney, respectively, in piglets,⁷¹ and the total number of small intestinal cells is much larger than liver and kidney cells.⁷³ The main consumer of citrulline is the kidney. In a study by Windmueller et al.,⁷⁴ citrulline utilization was measured in isolated livers perfused for 150 min with blood-plus-plasma. A tracer dose of L-[carbamoyl-14C] citrulline was added to the recycling perfusate, which contained 124 µM citrulline. After 150 min and about 40 passes through, about 90% of the labeled citrulline remained, which indicates that the liver is very inefficient in metabolizing citrulline. In contrast, 35% of citrulline is consumed by arterial blood as it passes through the kidney.^74,75 Nowadays, the small intestinal absorptive epithelium is widely regarded as the main source of circulating citrulline.

In several organ exclusion experiments, it was observed that no part of the body, except the intestine, releases large amounts of citrulline under physiological conditions.⁷⁴ The use of specific inhibitors of small intestinal OAT⁶⁶ and OCT⁷⁶ for small bowel targeting intervention significantly reduced plasma citrulline concentrations, which can also support this conclusion.

Experimental and clinical data⁷⁷ have shown an uneven distribution of citrulline production within the small intestine. It was observed that the P5CS activity of rats is distributed in the duodenum, upper jejunum, lower jejunum, and ileum at 26%, 31%, 33%, and 10%, respectively.⁶⁹ However, the data provided by Crenn et al.^78,79 indicate that there is a volume effect. The decrease in intestinal absorption after irradiation is associated with the loss of functionally active intestinal epithelial cells that make up the surface of the absorbable mucosa.^80

-83

Plasma citrulline concentration and the radiation dose

The relationship between the plasma citrulline concentration and the radiation dose was proven many times through experiments and clinical studies. Lutgens et al⁸⁴ used female NMRI mice to investigate the relationship between plasma citrulline levels and X-ray-induced small intestine epithelial cell loss and small intestinal morphology. The conclusion is that the plasma citrulline concentration changes most significantly at the time points of 84 hours and 4 days after IR. At low doses (0-3 Gy) the plasma citrulline concentration changes, although it is not obvious, but at a high dose (3-12 Gy), the decline is obvious. After the fourth day, the citrulline level began to recover and reached normal level in mice that received less than 8 Gy irradiation, while mice that received higher doses of irradiation were not able to fully recover. Lutgens et al⁸⁵ conducted a prospective clinical study in patients undergoing graded radiotherapy for abdominal and/or pelvic cancer sites (23 patients, 9 males, 14 females, 28.3-72.6 years old). After the initiation of radiotherapy, the citrulline concentration showed a decrease in relation to the dose received and the volume of the intestine. The citrulline concentration in the last 3 weeks of treatment showed correlation with evaluated clinical toxicity. The acid concentration showed more relevant correlation with the dose or exposure volume than with the evaluated clinical toxicity. From November 2008 to May 2010, 53 patients (36 prostate cancer, 17 endometrial cancer) who underwent pelvic radiation therapy were prospectively reviewed in Turkey.³⁰ A strong correlation between dose-volume and citrulline concentration was also observed, and the authors recommend that citrulline concentration should be included as an indicator of intestinal toxicity caused by radiation in future clinical practice. The relationship between citrulline concentration and intestinal epithelial cell loss is also observed in other pathological conditions not related to radiation, such as surgery after small bowel transplantation,^78,79,86,87 celiac disease and non-Celiac disease,⁸⁸ and viral enteritis.⁸⁹ Overall, plasma citrulline appears to be a quantitative parameter and it is not depend on related to the underlying cause of epithelial cell loss.⁹⁰

Non-Radiation Factors Affecting Plasma Citrulline Concentration

Crenn⁹¹ examined several non-IR factors that affect plasma citrulline concentrations, including diet, age and ethnicity, renal function, metabolic stress and inflammation, and liver function. Recently, a study using 3 types of animals (mice, minipigs, and Rhesus macaques)⁹² found that the citrulline level was significantly reduced by 35.5% (P < 0.0017), when nonhuman primates (NHPs) anesthetized with ketamine and acepromazine compared with unanesthetized NHPs. It is also found that in the postprandial state, the concentration of citrulline in NHPs decreased slightly, but decreased significantly by 12.2%. These results indicate that plasma citrulline is affected by experimental conditions such as anesthesia and feeding. In a study by Park et al⁹³ it was found that serum citrulline levels in mice showed diurnal changes and fluctuations related to food intake with no significant simultaneous change in the intestinal cell mass. Serum citrulline levels in fed mice did not change daily, while in fasted mice it was significantly higher in the morning than at night. These findings highlight the importance of consistency in sample collection strategies in translational research.

Citrulline in Non-Plasma Body Fluids

Because blood extraction can cause damage to the body, blood extraction and storage require professional skills; thus, it is not the most suitable method for large-scale screening of radiation-exposed individuals. In addition to detecting citrulline in plasma, changes in citrulline concentrations have also been detected in urine and saliva after exposure to radiation. In an experiment in which 3 male and 4 female rhesus monkeys (Macaca mulatta) were irradiated with cobalt 60⁹⁴ at the dose of 4 Gy, saliva was collected at different time points and citrulline levels were determined by an ultra-high performance liquid chromatography system in combination with Xevo G2-S time-of-flight mass spectroscopy (TOF-MS). The results showed that on the first day after irradiation, the citrulline in saliva increased rapidly to more than 2 times of that before the irradiation, and then rapidly decreased again on the third day. This result is the exact opposite of how plasma citrulline responds to radiation (which drops significantly in blood). In C57BL/6 mice, the γ-ray (cesium 137) irradiation dose was 0.5-8 Gy, and the sampling time was 1 and 7 days after irradiation. No change in citrulline concentration was found in saliva.⁹⁵ Although saliva is easy to obtain, as a biomarker its application has many restrictions because of factors such as smoking,⁹⁶ circadian rhythm,⁹⁷ eating habits,⁹⁸ and so on. In some pathological situations, saliva contains certain blood components, which may affects the results.⁹⁹

Goudarzi and colleagues¹⁰⁰ applied different radiation patterns to C57BL/6 mice, and compared the effects of internal (Sr 90 and Cs 137) and external irradiation (low and high dose rates of X-rays). The results showed that there was no statistically significant change in urinary citrulline concentration within 24 hours after 4.4 Gy irradiation with X-rays at a low dosing rate (3.0 mGy/min). The citrulline levels in mice were significantly reduced after 90 days of 90Sr (internal irradiation) exposure with a cumulative dose of 2.0 Gy, and Cs 137 (internal irradiation) on the fifth day after exposure with a cumulative dose of 4.1 Gy also showed a similar trend. After X-ray irradiation with 4.4 Gy at a high dosing rate (1.1 Gy/min), the citrulline level on the 5th day increased significantly. The results of this study are very important. It illustrates that changes in citrulline can be detected under internal radiation, and inhalation internal radiation occurs in many nuclear accidents. In several other investigations involving nontargeted mass spectrometry detection of sources other than irradiation, the change in citrulline was not detected in radiation-exposed mice,¹⁰¹ rats,¹⁰² and monkeys.¹⁰³ This may be due to the nontargeted approach used. It can be concluded from the above researches that the level of citrulline in body fluid can directly represent neither the change of citrulline concentration in plasma nor the loss of small intestinal epithelial cells.

Conclusions

There are different types of radiation in various scenarios, and they affect different groups of people (classified based on protection level, gender, type of radiation exposure, age, education level, etc.). In the case of a certain group size, appropriate measures should be taken. This calls for a targeted approach, which can combine clinical symptoms and biomarkers to achieve the current optimal solution. The currently used methods aim to find a marker that meets all conditions. However, the reality is that in different biological processes, biomolecules that can play a role in marking are often different. Thus different biomarkers should be used in different biological and clinical symptom stages, and radiation-induced tissue damage cannot be expressed or quantified by a single functional or morphological parameter.¹⁰⁴

In addition, as blood citrulline is being increasingly accepted for its high-dose external radiation, clinicians are hopeful that the concentration of citrulline can be consistent with the current clinical toxicity classification system. If clinical toxicity classification and radiation biomarkers can be mapped with clear biological processes and clinical symptoms, it will be greatly beneficial to the decision-making process.

Footnotes

Authors’ Note

Fei Ye and Jing Ning contributed equally. The administrative support of Ms. Hiromi Arai, Ms. Mikiko Nakajima, Ms. Chianing Hsieh, Ms. Yasuko Morimoto, and Ms. Kaoru Tanaka, is gratefully acknowledged. Thanks are also due to the anonymous peer reviewers for providing the constructive comments that strengthened the presentation of this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interests with respect to the research, authorship, and publication of this paper.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The third author (Zeenath Fardous) was a fellow of the Nuclear Researchers Exchange Programme 2018 supported by the Ministry of Education, Culture, Sport, Sciences and Technology (MEXT), Japan, and the Nuclear Safety Research Association, Japan. This work was partially supported by MEXT Grant-in-Aid for Scientific Research on Innovative Areas “Living in Space” (Grant Numbers: 15H05935, 15K21745).

ORCID iD

Zeenath Fardous

Takanori Katsube

Bing Wang

References

Lacombe

Sima

Amundson

Zenhausern

Candidate gene biodosimetry markers of exposure to external ionizing radiation in human blood: a systematic review. PLoS One. 2018;13(6):e0198851 .

Dubois

Walker

. Prospects for management of gastrointestinal injury associated with the acute radiation syndrome. Gastroenterology. 1988;95(2):500–507.

Perez

Grigsby

Lockett

Chao

Williamson

. Radiation therapy morbidity in carcinoma of the uterine cervix: dosimetric and clinical correlation. Int J Radiat Oncol Biol Phys. 1999;44(4):855–866.

Miller

Martenson

Nelson

, et al. The incidence and clinical consequences of treatment-related bowel injury. Int J Radiat Oncol Biol Phys. 1999;43(4):817–825.

Martin

Pointreau

Roche-Forestier

Barillot

. Normal tissue tolerance to external beam radiation therapy: small bowel. Cancer Radiother. 2010;14(4-5):350–353.

Emami

Lyman

Brown

, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys. 1991;21(1):109–122.

Shadad

Sullivan

Martin

Egan

. Gastrointestinal radiation injury: symptoms, risk factors and mechanisms. World J Gastroenterol. 2013;19(2):185–198.

Qiu

Roberts

Potten

. Cell migration in the small and large bowel shows a strong circadian rhythm. Epithelial Cell Biol. 1994;3(4):137–148.

Hall

Giaccia

AJ.

Radiobiology for the Radiologist. 7th ed. Atithi Medical Books Pvt Ltd; 2012.

10.

Cheema

Mehta

Fatanmi

, et al. A metabolomic and lipidomic serum signature from nonhuman primates administered with a promising radiation countermeasure, gamma-tocotrienol. Int J Mol Sci. 2017;19(1):79.

11.

Chinsoo Cho

L GE

. Radiation injury. In: Fauci

Isselbacher

Braunwald

, et al. (eds) Harrison’s Principles of Internal Medicine. New York, NY: McGraw Hill; 1998:2559.

12.

MacVittie

Farese

Bennett

, et al. The acute gastrointestinal subsyndrome of the acute radiation syndrome: a rhesus macaque model. Health Phys. 2012;103(4):411–426.

13.

Vigneulle

Rao

Fasano

MacVittie

. Structural and functional alterations of the gastrointestinal tract following radiation-induced injury in the rhesus monkey. Dig Dis Sci. 2002;47(7):1480–1491.

14.

Potten

. Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci. 1998;353(1370):821–830.

15.

Marshman

Booth

Potten

. The intestinal epithelial stem cell. BioEssays. 2002;24(1):91–98.

16.

Gaugler

Neunlist

Bonnaud

Aubert

Benderitter

Paris

. Intestinal epithelial cell dysfunction is mediated by an endothelial-specific radiation-induced bystander effect. Radiat Res. 2007;167(2):185–193.

17.

Suzuki

Yamashita

Radiation-induced bystander response: mechanism and clinical implications. Adv Wound Care. 2014;3(1):16–24.

18.

Goldberg

Lehnert

. Radiation-induced effects in unirradiated cells: a review and implications in cancer. Int J Oncol. 2002;21(2):337–349.

19.

Morgan

WF.

Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro. Radiat Res. 2003;159(5):567–580.

20.

Stansborough

Bateman

Al-Dasooqi

, et al. Fractionated abdominal irradiation induces intestinal microvascular changes in an in vivo model of radiotherapy-induced gut toxicity. Support Care Cancer. 2017;25(6):1973–1983.

21.

Blutt

Klein

Donowitz

Shroyer

Guha

Estes

. Use of organoids to study regenerative responses to intestinal damage. Am J Physiol Gastrointest. 2019;317(6):G845–G852.

22.

Sato

Vries

Snippert

, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262–265.

23.

Leonetti

Estephan

Ripoche

, et al. Secretion of acid sphingomyelinase and ceramide by endothelial cells contributes to radiation-induced intestinal toxicity. Cancer Res. 2020;80(12):2651–2662.

24.

Saha

Bhanja

Liu

, et al. TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome. PLoS One. 2012;7(1):e29357.

25.

Solanas

Batlle

. Control of cell adhesion and compartmentalization in the intestinal epithelium. Exp Cell Res. 2011;317(19):2695–2701.

26.

Huynh

Akcora

Malaterre

, et al. CSF-1 receptor-dependent colon development, homeostasis and inflammatory stress response. PLoS One. 2013;8(2):e56951.

27.

Vanuytsel

Senger

Fasano

Shea-Donohue

. Major signaling pathways in intestinal stem cells. Biochim Biophys Acta. 1830(2):2410–2426.

28.

Potten

Loeffler

. A comprehensive model of the crypts of the small-intestine of the mouse provides insight into the mechanisms of cell-migration and the proliferation hierarchy. J Theor Biol. 1987;127(4):381–391.

29.

Schofield

. The stem-cell system. Biomed Pharmacother. 1983;37(8):375–380.

30.

Onal

Kotek

Unal

, et al. Plasma citrulline levels predict intestinal toxicity in patients treated with pelvic radiotherapy. Acta Oncologica. 2011;50(8):1167–1174.

31.

Blijlevens

. Cytotoxic treatment-induced gastrointestinal symptoms. Curr Opin Support Pa. 2007;1(1):16–22.

32.

Wardill

Bowen

. Chemotherapy-induced mucosal barrier dysfunction: an updated review on the role of intestinal tight junctions. Curr Opin Support Pa. 2013;7(2):155–161.

33.

Bellm

Epstein

Rose-Ped

Martin

Fuchs

. Patient reports of complications of bone marrow transplantation. Supportive Care Cancer. 2000;8(1):33–39.

34.

Powell

. Barrier function of epithelia. Am J Physiol. 1981;241(4): G275–288.

35.

Barzal

Szczylik

Rzepecki

Jaworska

Anuszewska

. Plasma citrulline level as a biomarker for cancer therapy-induced small bowel mucosal damage. Acta Biochim Pol. 2014;61(4):615–631.

36.

Treister

Sonis

. Mucositis: biology and management. Curr Opin Otolaryngol Head Neck Surg. 2007;15(2):123–129.

37.

Sonis

. The pathobiology of mucositis. Nature Rev Cancer. 2004;4(4):277–284.

38.

Peterson

Bensadoun

Roila

Grp

EGW

. Management of oral and gastrointestinal mucositis: ESMO Clinical Practice Guidelines. Ann Oncol. 2011;22(6):vi78–vi84.

39.

Van Vliet

Harmsen

de Bont

Tissing

. The role of intestinal microbiota in the development and severity of chemotherapy-induced mucositis. PLoS Pathogens. 2010;6(5):e1000879.

40.

Gate

Paul

Tew

Tapiero

. Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother. 1999;53(4):169–180.

41.

Sonis

. The biologic role for nuclear factor-kappaB in disease and its potential involvement in mucosal injury associated with anti-neoplastic therapy. Crit Rev Oral Biol Med. 2002;13(5):380–389.

42.

Maddens

Charruyer

Plo

, et al. Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect. Blood. 2002;100(4):1294–1301.

43.

Dorr

Emmendorfer

Haide

Kummermehr

. Proliferation equivalent of accelerated repopulation in mouse oral-mucosa. Int J Radiat Biol. 1994;66(2):157–167.

44.

Sonis

Peterson

Edwards

, et al. Defining mechanisms of action of interleukin-11 on the progression of radiation-induced oral mucositis in hamsters. Oral Oncol. 2000;36(4):373–381.

45.

D’Agostino

Ciacci

Capuano

, et al. Metabolic fate of plasma diamine oxidase: evidence of isolated and perfused rat liver uptake. Digestion. 1986;34(4):243–250.

46.

Bieganski

Kusche

Lorenz

Hesterberg

Stahlknecht

Feussner

. Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism. Biochim Biophys Acta. 1983;756(2):196–203.

47.

Bragg

Thompson

West

. Intestinal diamine oxidase levels reflect ischemic injury. J Surg Res. 1991;50(3):228–233.

48.

Bounous

Echave

Vobecky

Navert

Wollin

. Acute necrosis of the intestinal mucosa with high serum levels of diamine oxidase. Digest Dis Sci. 1984;29(9):872–874.

49.

Wolvekamp

de Bruin

. Diamine oxidase: an overview of historical, biochemical and functional aspects. Digest Dis. 1994;12(1):2–14.

50.

Tsunooka

Maeyama

Hamada

, et al. Bacterial translocation secondary to small intestinal mucosal ischemia during cardiopulmonary bypass. Measurement by diamine oxidase and peptidoglycan. Eur J Cardiothorac Surg. 2004;25(2):275–280.

51.

Costa

Mumolo

Bellini

, et al. Role of faecal calprotectin as non-invasive marker of intestinal inflammation. Digest Liver Dis. 2003;35(9):642–647.

52.

D’Haens

Ferrante

Vermeire

, et al. Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis. 2012;18(12):2218–2224.

53.

Manceau

Chicha-Cattoir

Puy

Peoch

. Fecal calprotectin in inflammatory bowel diseases: update and perspectives. Clin Chem Lab Med. 2017;55(4):474–483.

54.

Summerton

Longlands

Wiener

Shreeve

. Faecal calprotectin: a marker of inflammation throughout the intestinal tract. Eur J Gastroen Hepat. 2002;14(8):841–845.

55.

Reis Ferreira

Andreyev

HJN

Mohammed

, et al. microbiota- and radiotherapy-induced gastrointestinal side-effects (mars) study: a large pilot study of the microbiome in acute and late-radiation enteropathy. Clin Cancer Res. 2019;25(21):6487–6500.

56.

Liu

Zhou

Wang

, et al. impact of low-dose ionising radiation on the composition of the gut microbiota of mice. Toxicol Sci. 2019;171(1):258–268.

57.

Crawford

Gordon

. Microbial regulation of intestinal radiosensitivity. Proc Natl Acad Sci U S A. 2005;102(37):13254–13259.

58.

Lam

Moulder

Salzman

Dubinsky

Andersen

Baker

. Intestinal microbiota as novel biomarkers of prior radiation exposure. Radiat Res. 2012;177(5):573–583.

59.

Broin

PÓ

Vaitheesvaran

Saha

, et al. Intestinal microbiota-derived metabolomic blood plasma markers for prior radiation injury. Int J Radiat Oncol Biol Phys. 2015;91(2):360–367.

60.

Goudarzi

Mak

Jacobs

, et al. An integrated multi-omic approach to assess radiation injury on the host-microbiome axis. Radiat Res. 2016;186(3):219–234.

61.

Nam

Kim

Seo

Kang

Bae

. Impact of pelvic radiotherapy on gut microbiota of gynecological cancer patients revealed by massive pyrosequencing. PLoS One. 2013;8(12):e82659.

62.

Manichanh

Varela

Martinez

, et al. The gut microbiota predispose to the pathophysiology of acute postradiotherapy diarrhea. Am J Gastroenterol. 2008;103(7):1754–1761.

63.

Wang

Ling

Yang

, et al. Gut microbial dysbiosis may predict diarrhea and fatigue in patients undergoing pelvic cancer radiotherapy: a pilot study. PLoS One. 2015;10(5):e0126312.

64.

Kumagai

Rahman

Smith

. The microbiome and radiation induced-bowel injury: evidence for potential mechanistic role in disease pathogenesis. Nutrients. 2018;10(10):1405.

65.

Knabe

Flynn

. Synthesis of Citrulline from Glutamine in Pig Enterocytes. Biochem J. 1994;299(1):115–121.

66.

Flynn

. An important role for endogenous synthesis of arginine in maintaining arginine homeostasis in neonatal pigs. Am J Physiol-Reg I. 1996;271(5):R1149–R1155.

67.

Wakabayashi

Henslee

Jones

. Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal-mucosa—subcellular-localization and temperature stability. J Biol Chem. 1983;258(6):3873–3882.

68.

Kramer

Henslee

Wakabayashi

Jones

. Delta-1-pyrroline-5-carboxylate synthase from rat intestinal-mucosa. Method Enzymol. 1985;113:113–120.

69.

Wakabayashi

Yamada

Hasegawa

Yamada

. Enzymological evidence for the indispensability of small-intestine in the synthesis of arginine from glutamate .1. Pyrroline-5-carboxylate synthase. Arch Biochem Biophys. 1991;291(1):1–8.

70.

Davis

Flynn

Knabe

Davidson

. Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs. J Nutr. 1997;127(12):2342–2349.

71.

Samuels

Aarts

HLM

Ball

. Effect of dietary proline on proline metabolism in the neonatal pig. J Nutr. 1989;119(12):1900–1906.

72.

Knabe

. Arginine synthesis in enterocytes of neonatal pigs. Faseb J. 1995;9(4): A742–A742.

73.

Schoknecht

Pond

. Short-term ingestion of a high protein diet increases liver and kidney mass and protein accretion but not cellularity in young pigs. Proc Soc Exp Biol Med By. 1993;203(2):251–254.

74.

Windmueller

Spaeth

. Source and fate of circulating citrulline. Am J Physiol. 1981;241(6): E473–480.

75.

Dejong

Welters

Deutz

Heineman

Soeters

. Renal arginine metabolism in fasted rats with subacute short bowel syndrome. Clin Sci. 1998;95(4):409–418.

76.

Hoogenraad

Totino

Elmer

Wraight

Alewood

Johns

. Inhibition of intestinal citrulline synthesis causes severe growth-retardation in rats. Am J Physiol. 1985;249(6): G792–G798.

77.

Van der Hulst

von Meyenfeldt

Deutz

Soeters

. Glutamine extraction by the gut is reduced in depleted [corrected] patients with gastrointestinal cancer. Annal Surg. 1997;225(1):112–121.

78.

Crenn

Coudray-Lucas

Thuillier

Cynober

Messing

. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterol. 2000;119(6):1496–1505.

79.

Chang

Javid

, et al. Serial transverse enteroplasty enhances intestinal function in a model of short bowel syndrome. Annal Surg. 2006;243(2):223–228.

80.

Overgaard

Matsui

. Effect of radiation on glucose absorption in the mouse jejunum in vivo. Radiother Oncol. 1990;18(1):71–77.

81.

Juby

Dixon

Axon

. Abnormal intestinal permeability and jejunal morphometry. J Clin Pathol. 1987;40(7):714–718.

82.

Gunter-Smith

PJ.

Gamma radiation affects active electrolyte transport by rabbit ileum. II. Correlation of alanine and theophylline response with morphology. Radiat Res. 1989;117(3):419–432.

83.

Kirichenko

Mason

Straume

Teates

Rich

. Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction. Radiat Res. 2000;153(2):164–172.

84.

Lutgens

Deutz

Gueulette

, et al. Citrulline: a physiologic marker enabling quantitation and monitoring of epithelial radiation-induced small bowel damage. Int J Radiat Oncol Biol Phys. 2003;57(4):1067–1074.

85.

Lutgens

Deutz

Granzier-Peeters

, et al. Plasma citrulline concentration: a surrogate end point for radiation-induced mucosal atrophy of the small bowel. A feasibility study in 23 patients. Int J Radiat Oncol Biol Phys. 2004;60(1):275–285.

86.

Rhoads

Plunkett

Galanko

, et al. Serum citrulline levels correlate with enteral tolerance and bowel length in infants with short bowel syndrome. J Pediatr. 2005;146(4):542–547.

87.

Jianfeng

Weiming

Ning

, et al. Serum citrulline is a simple quantitative marker for small intestinal enterocytes mass and absorption function in short bowel patients. J Surg Res. 2005;127(2):177–182.

88.

Crenn

Vahedi

Lavergne-Slove

Cynober

Matuchansky

Messing

Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterol. 2003;124(5):1210–1219.

89.

Gondolesi

Fishbein

Chehade

, et al. Serum citrulline is a potential marker for rejection of intestinal allografts. Transplant Proc. 2002;34(3):918–920.

90.

Lutgens

Lambin

Biomarkers for radiation-induced small bowel epithelial damage: an emerging role for plasma citrulline. World J Gastroentero. 2007;13(22):3033–3042.

91.

Crenn

Messing

Cynober

. Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction. Clin Nutr. 2008;27(3):328–339.

92.

Bujold

Hauer-Jensen

Donini

, et al. Citrulline as a biomarker for gastrointestinal-acute radiation syndrome: species differences and experimental condition effects. Radiat Res. 2016;186(1):71–78.

93.

Park

Shaughnessy

Armenia

Cowles

. Serum citrulline levels exhibit circadian variation and fluctuations in relation to food intake in mice. Gastroenterol Res. 2019;12(2):88–92.

94.

Laiakis

Nishita

Bujold

, et al. Salivary metabolomics of total body irradiated nonhuman primates reveals long-term normal tissue responses to radiation. Int J Radiat Oncol Biol Phys. 2019;105(4):843–851.

95.

Laiakis

Strawn

Brenner

Fornace

Assessment of saliva as a potential biofluid for biodosimetry: a pilot metabolomics study in mice. Radiat Res. 2016;186(1):92–97.

96.

Rathnayake

Akerman

Klinge

, et al. Salivary biomarkers for detection of systemic diseases. PLoS One. 2013;8(4): e61356.

97.

Papagerakis

Zheng

Schnell

, et al. The circadian clock in oral health and diseases. J Dent Res. 2014;93(1):27–35.

98.

Goodson

Kantarci

Hartman

, et al. Metabolic disease risk in children by salivary biomarker analysis. PLoS One. 2014;9(6): e98799.

99.

Kivlighan

Granger

Schwartz

. Blood contamination and the measurement of salivary progesterone and estradiol. Horm Behav. 2005;47(3):367–370.

100.

Goudarzi

Chauthe

Strawn

Weber

Brenner

Fornace

. Quantitative metabolomic analysis of urinary citrulline and calcitroic acid in mice after exposure to various types of ionizing radiation. Int J Mol Sci. 2016;17(5);782.

101.

Chen

Brenner

Brown

. Identification of urinary biomarkers from X-irradiated mice using NMR spectroscopy. Radiat Res. 2011;175(5):622–630.

102.

Lanz

Patterson

Slavik

, et al. Radiation metabolomics. 3. Biomarker discovery in the urine of gamma-irradiated rats using a simplified metabolomics protocol of gas chromatography-mass spectrometry combined with random forests machine learning algorithm. Radiat Res. 2009;172(2):198–212.

103.

Johnson

Patterson

Krausz

, et al. Radiation metabolomics. 5. Identification of urinary biomarkers of ionizing radiation exposure in nonhuman primates by mass spectrometry-based metabolomics. Radiati Res. 2012;178(4):328–340.

104.

Griffiths

. The example of gastrointestinal damage induced by ionising radiation: are there accessible markers? Cell Mol Biol. 2001;47(3):427–435.

Citrulline,A Potential Biomarker of Radiation-Induced Small Intestine Damage

Abstract

Keywords

Introduction

Radiation Damage of the Small Intestine

Clinical Symptoms of Small Intestinal Damage Caused by Radiation

Molecular Mechanism of Small Intestine Damage Caused by Radiation

Biomarkers of Radiation Damage in the Small Intestine

Citrulline as Biomarkers of Radiation Damage in the Small Intestine

Features of citrulline secretion in the small intestine

Plasma citrulline concentration and the radiation dose

Non-Radiation Factors Affecting Plasma Citrulline Concentration

Citrulline in Non-Plasma Body Fluids

Conclusions

Footnotes

Authors’ Note

Declaration of Conflicting Interests

Funding

ORCID iD

References