ABSTRACT :

Radionuclides with a long half-life such as that of K-40 have existed on Earth for 4.6 billion years (By), since its very origin. Some radionuclides, such as H-3 and C-14, are formed continuously in the atmosphere. These are in fact essential body components: all organisms are radioactive and are constantly and invariably exposed to ionizing radiation (IR) internally and externally. Humans are estimated to receive approximately 20,000 radiation impacts each second. Because living organisms have been exposed to IR throughout their evolutional history of 4 By, their intrinsic nature is to respond and adapt dynamically to environmental factors. The effects of IR are actually integrated into their genomes. A huge number of microbes use \text{H}_2, a product of radiolysis of water, as an energy source. Organisms use reactive oxygen species (ROS), major products of action of IR, as signaling agents. Numerous and diverse species show radiation hormesis: stimulation at low doses and inhibition at higher doses. Hormesis has beneficial effects: most atomic bomb survivors have exhibited long life and reduced cancer mortality, although the small fraction of people exposed to higher doses received no hormetic benefit. When organisms were placed under low-IR conditions, with shielding by lead bricks, put deep underground, and fed with K-40 deficient food, their activity was markedly reduced. Later, they recovered because of supplemental irradiation, strongly suggesting that IR is indispensable for normal life. IR apparently supports basic biochemical reactions. Judging from these findings, the linear no-threshold model (LNT), a hypothesis which holds that even the smallest amount of radiation is harmful, is invalid.

Keywords: Atomic Bomb Survivors; Hormesis; Ionizing Radiation; Life Span Study; Linear No-threshold Model.

Abbreviations

BEAR I: Biological Effects of Atomic Radiation Committee
By: Billion Years
ICRP: International Commission on Radiological Protection
IR: Ionizing Radiation
LNT: Linear No-threshold
LDR: Low Dose Radiation
LSS: Life Span Study
NAS: National Academy of Sciences
NIC: Not-in-the-city control
RF: Rockefeller Foundation
ROS: Reactive Oxygen Species

1. INTRODUCTION

In the aftermath of the Great East Japan Earthquake of March 11, 2011, residents in a radius of 20 km from the Fukushima Daiichi nuclear power plant were forced to evacuate. Their tentative home-return program was conducted during May–September, during which they were allowed to visit their homes for two hours to gather important and valuable items. For about a week in mid-July, the author engaged in measurement of their radio-contamination using a Geiger–Muller counter (Sutou, 2015). On this occasion, radioactivity was measured at various places in Fukushima and in other prefectures; the highest count was 6,000 cpm, which was personally unsettling because most people had been taught that the smallest amount of radiation is harmful, as asserted on the basis of the linear no-threshold model (LNT). Thereafter, the author personally scrutinized the effects of ionizing radiation (IR) on living organisms. An analysis of the collected findings indicates that the LNT is invalid. By stark contrast, low-dose radiation (LDR) is both indispensable and beneficial.

2. WE LIVE AMID IONIZING RADIATION (IR): ALL LIVING ORGANISMS ARE RADIOACTIVE

1.1. Relations between ionizing radiation and life
Figure 1 presents a brief illustration of the relation between IR and life (Sutou, 2022). Living organisms have lived amid radionuclides and have continued to be exposed to IR throughout the evolutional history of 4 By: all organisms are radioactive. In fact, all have integrated the effects of IR into their respective genomes. A huge number of microbes live on \text{H}_2, a radiolysis product. Moreover, IR produces reactive oxygen species (ROS), which organisms use as signaling agents and for antioxidation and detoxication. Organisms show hormesis; IR is not harmful, but it is in fact indispensable for life.
2.2. We live amid ionizing radiation (IR)
Radionuclides are abundant all around us (Table 1). Primary natural radionuclides with a long half-life have existed since the birth of the Earth 4.6 By ago. The absorption dose unit, Gy, is defined as one joule of radiation energy absorbed per kilogram of matter, signifying that radiation eventually produces heat. In fact, half of the heat of the Earth is supplied by the radiogenic decay of radionuclides (KamLAND, 2011). Although the half-lives of secondary natural radionuclides, the daughters of primary natural radionuclides, are short, they are produced continuously. Constantly, H-3 and C-14, which are induced radionuclides generated in the air by cosmic rays, are taken into living organisms. A typical artificial radionuclide is F-18, which is used for PET diagnosis.
3.3. Biological effects of ionizing radiation (IR) studied during the Manhattan project
Atomic bombs were produced under the Manhattan project, during which the biological effects of IR were also investigated. The chief of the Drosophila study was Stern of the University of California, Berkeley. He invited Muller as an adviser, actually for work as the practical leader.
Of several studies, the summaries of two studies using low-dose \gamma rays are presented in Table 4. Caspari’s results indicated the existence of a threshold (Caspari and Stern, 1948), whereas Uphoff’s data showed no threshold (Uphoff and Stern, 1949). The statistical significance reported from the Uphoff study was mainly attributable to the low mutation rate of the negative control. Likely under Muller’s leadership, Stern rejected Caspari, a skilled scientist, falsely accusing him of sampling errors, resulting in high negative control data, which were actually normal and close to the expected value of 0.25%. Instead, Stern supported Uphoff, a postgraduate student who joined Stern’s laboratory after Caspari’s departure, even though Uphoff’s control data were unusually low. Stern’s promise to publish Uphoff’s study later was broken. Some experimental or theoretical failures might be involved in her study, e.g., the control flies had been mistakenly irradiated with leaked \gamma rays (Calabrese et al., 2022).

Atomic bombs were dropped on Hiroshima and Nagasaki in 1945. In 1946, Muller was awarded a Nobel Prize. He knew Caspari’s data (Table 4) and recognized their importance. He contradicted Caspari in his Nobel lecture and insisted that no threshold in genetic effects of IR exists (Muller, 1946). The idea of the “proportionality rule” or LNT propagated widely thereafter.
3.4. Close relation between Muller and Rockefeller Foundation
The Rockefeller Foundation (RF) was established in 1914 by John Rockefeller, founder of Standard Oil Co. A main objective was to contribute to human welfare. To achieve this, RF emphasized eugenics. Muller was an eager supporter of eugenics. To achieve the goals of eugenics, he emphasized genetics. Morgan, an authority of genetics, and Muller were at Columbia University. RF had the means to keep in touch with Muller and make him one of the RF brains. In fact, RF had fully supported Muller, probably from around 1914 until his death in 1967. Reportedly, support by RF influenced Muller’s selection for a Nobel Prize.
3.5. Recommendation of linear no-threshold model (LNT) by the US National Academy of Sciences (NAS)
Nuclear energy had remained entirely under the control of military forces during 1946–1953, after which US President Eisenhower served for eight years. He made the speech extolling “Atoms for peace,” at the United Nations on December 8, 1953, and liberated nuclear energy for use by the general public. In 1954, RF financed NAS to establish the Genetics Panel of the Biological Effects of Atomic Radiation Committee (BEAR I) in NAS, the president of which was D. Bronk, also the president of both Rockefeller Institute and Rockefeller University and an RF Board member. Moreover, RF appointed mathematician W. Weaver, who had been the director of RF’s Division of Natural Sciences since 1932 and who was in charge of funding. Muller was pivotal among the 16 BEAR I members. Importantly, RF provided key members with funds.
On June 12, 1956, BEAR I published its report, “Genetic effects of atomic radiation” declaring that no safe dose of radiation exists (BEAR I, 1956). The next day, the New York Times (NYT) reported on its front page, “The future of mankind was in danger. Scientists term radiation a peril to future of man. Even small dose can prove harmful to descendants of victim.” A.H. Sulzberger, the NYT publisher and an RF Board member, facilitated the publication of the BEAR I report. Most mass media followed the news.
This report is problematic (Calabrese, 2022). 1) Questionable data of fruit flies were fundamentally used (Fig. 3A; Table 4). 2) Muller rejected Neel’s proposal (Neel and Schull, 1956) to use data indicating that there were no genetic effects in offspring of atomic bomb study participants. 3) The article is authored anonymously. 4) Data for abnormalities that were estimated by nine members were manipulated arbitrarily, which decreased the variation and raised the average of the data. 5) The manuscript was written by a journal writer and was not circulated among BEAR I members.
Because the BEAR I report was published under the sponsorship of NAS, the highest authority of the scientific world, it led to huge effects thereafter. 1) A threshold of 2 mGy/d set by the International Commission on Radiological Protection (ICRP) in 1934 was replaced with LNT. 2) NAS expanded LNT to cancer risk estimation (NAS, 2006), which Sutou (2018) criticized. 3) LNT has remained the basis of radiation regulation to the present day (ICRP, 2007).

4. RADIATION HORMESIS IS PREVALENT

4.1. Living organisms respond to ionizing radiation (IR)
The intrinsic nature of living organisms is to respond to chemicals (e.g., nutrients and toxins), physical conditions (e.g., temperature and humidity), and other organisms (e.g., prey and predators) in the environment. Regarding IR, living organisms can sense IR by detecting its products such as ROS and DNA damage.
Luckey (2019) lists 1,269 hormesis-associated references, which include broadly diverse species: viruses, bacteria, fungi, yeasts, algae, plants, protozoa, invertebrates, and vertebrates. Calabrese and Blain (2005) produced a hormesis database using dose responses of 1,654 types of radiation and radionuclides (29%), 1308 inorganic compounds (23%), and organic compounds (47%). Many species Luckey (2019) listed are included. It can be said that radiation and chemical hormesis are ubiquitous among living organisms. A PubMed search of “hormesis, hormetic response, or adaptive response” yielded 202,989 articles as of February 17, 2023.
4.2. Some typical hormetic responses
Because radiation hormesis is ubiquitous among living organisms, some examples can be shown (Fig. 4). The growth of Tetrahymena is stimulated by \gamma rays, exhibiting hormesis (Fig. 4A). When \gamma rays were shielded with lead bricks, its growth was inhibited, hinting at the necessity of low-dose radiation for normal growth (indispensable region, Fig. 2). The mutation rate of Drosophila irradiated with X rays showed a dose-response U-shaped curve, which is a hallmark of hormesis (Fig. 4B; Fig. 3B). The lifespan of mice irradiated with \gamma rays was elongated by lower dose rates, but shortened by higher ones (Fig. 4C). Granted that the natural background radiation is .4 mGy/y, 1–10 mGy/d corresponds to 132–1,320 times of the background. When the relation between radon levels and lung cancer incidence was examined, the dose-response curve showed a reverse correlation, utterly negating LNT (Fig. 4D). Higher incidence of lung cancer was observed at lower radon levels (Fig. 4D, below the thick arrows). Some radon exposure seems to indicate prevention of lung cancer (hormetic region, Fig. 2).
4.3. Hormesis seen among Hiroshima and Nagasaki A-bomb survivors
On August 6, 1945, an atomic bomb was dropped on Hiroshima. The energy consisted of 50% blast, 35% heat, and 15% radiation (Fig. 5A). Most people within 1 km radius of the epicenter must have been killed instantaneously by blast and heat, with a smaller number of people killed by radiation (Fig. 5B). Because the exposure dose estimation at the time was based only on the initial radiation (5%) and because residual radiation (10%) was neglected, the exposure doses were greatly underestimated.

5.1. Bacteria “eating” ionizing radiation (IR)

Desulforudis audaxviator bacteria live in a South African gold mine at 2.8-km depth (Chivian et al., 2008). As chemoautotrophic thermophiles, they obtain basic life sustenance from surrounding rocks using the radiation energy of U-238. Another radiation-eater was found in a 2-km-deep aquifer in eastern Siberia (Karnachuk et al., 2019).

5.2. Life thrives in deep subsurface areas with support of ionizing radiation (IR)

At the bottom of oceans, microbes in tightly packed sediments are able to survive by feeding off on organic matter imbedded in them or precipitated or deposited from the oceans above. Under sediments, basalt rocks appear and are followed by gabbro rocks above magma. These rock layers have pores and fissures into which seawater permeates. Along these cracks, biofilms are formed. They are a highly organized form of collective life, consisting of complex arrays of extracellular polymeric substances including polysaccharides, proteins, DNA, and small particles (Karygianni et al., 2020). Global cell numbers in biofilms mainly consisting of bacteria and archaea are estimated as 1.2 \times 10^{30} cells (Flemming and Wuertz, 2019). Biofilms might occupy 20–80% of the cells. Others are single sessile cells. Microbial cell densities in subseafloor basaltic rock can exceed 10^{10}\text{ cells/cm}^3 (Suzuki et al., 2020). In addition to bacteria and archaea, fungi are involved in biofilm formation (Nuppunen-Puputti et al., 2020).

Radiation supports thriving life in deep subsurface areas. A recent study indicated that water radiolysis produces H2 continuously. This has been found to be the primary electron donor (food) for microbes in continental aquifers kilometers below the earth’s surface (Sauvage et al., 2021). Microbes living in deeper areas depend more on radiolytic H2, which is produced particularly in rocky layers.

5.3. Low-dose radiation (LDR) is necessary: lead brick-shielding experiments

As portrayed in Fig. 4A, shielding radiation from \gamma rays with bricks retards the growth of Tetrahymena (Luckey, 1986). Similar results were obtained using the protozoan Paramecium tetraurelia and the cyanobacterium Synechococcus lividus (Planel et al., 1987). In addition to these organisms, the growth or viability of the following organisms was hindered: Mastigocladus laminosus (alga), Hordeum vulgare (barley), Artemia salina (brine shrimp), rats, and mice (Luckey, 2007).

5.4. Low dose radiation (LDR) is necessary: deep underground experiments

The underground Gran Sasso National Laboratory (LNGS), Italy, lies under thick rocks of about 1,300 m. At LNGS, cosmic rays and neutron fluxes are reduced by factors of 10^6 and 10^3, respectively, compared to a standard environment. The main constituents of the mountain ensure a low level of natural radioactivity, rendering it suitable for conducting biological experiments under low-radiation conditions. The dose rates of \gamma-rays measured using an organic scintillator at a ground control laboratory and LNGS were 357 \pm 16\text{ nGy/h} and 5.1 \pm 0.9\text{ nGy/h}, respectively. This indicates a reduction factor of approximately 70 in \gamma-ray activity at the LNGS compared to the control (Satta et al., 2002). Satta et al. (1995) observed an increased mutation rate when Saccharomyces cerevisiae (yeast) was cultured and treated with methyl methanesulfonate, mutagen, at LNGS. Similarly, Satta et al. (2002) found that mutation rates increased when Chinese hamster V79 cells were irradiated with \gamma-rays. If LNT is correct, then mutation rates must be lessened in low-radiation environments. These results suggest that DNA maintenance ability is hampered by low-radiation conditions.
The Waste Isolation Pilot Plant (WIPP, Carlsbad NM) is situated 650 m below the surface (Conca, 2017). WIPP is the sole repository in the U.S. for disposing of transuranic (TRU) nuclear waste, specifically waste generated from defense operations (EPA, 2023). This waste includes clothing, tools, rags, residues, debris, soil, and other items contaminated primarily with small amounts of \alpha-emitters like Pu-239, which require minimal shielding. Some of the waste contains \gamma-emitters, predominantly Cs-137 and Sr-90 isotopes resulting from the reprocessing of defense spent fuel. The waste is tightly packed in containers, and WIPP has efficient ventilation systems and maintains cleanliness. WIPP does not accept spent fuels from power plants, which have been kept in wet or dry storage. WIPP has the Low Background Radiation Experiment (LBRE) laboratory 960 m away from the waste disposal area. Deinococcus radiodurans, a radiation-resistant Gram-positive bacterium, was incubated in three environments: above-ground laboratory, LBRE laboratory, and a 6-inch pre-World War II steel chamber with estimated radioactivity levels of 1.75mGy/y, 0.0525 mGy/y and 0.0175 mGy/y respectively. Bacterial growth was monitored using protein levels, optical density, and cell agar plate counts, all of which indicated that higher radiation levels resulted in faster cell growth (Smith et al., 2011). Transcriptional analyses of this bacterium showed that background radiation is necessary for normal growth (Castillo et al., 2021). Growth retardation was also observed in V79 cells cultured at WIPP (Castillo et al., 2022).

5.5. Low dose radiation (LDR) is necessary: K-40 deficiency experiments

Of the 20,000 incident radiation hits humans receive every second, 4,500 originate from K-40 (Table 3). Luckey (2007) summarized the effects of a lack of K-40 on the growth of living organisms. Escherichia coli (bacterium), Tetrahymena pyriformis (protozoa), mice, and rats showed marked retardation of growth. Muckerheide (2000a) reports that mice fed with a diet lacking K-40 showed inferior growth, but a supplement of it recovered that growth. Publication of those findings was not done because the results were contradictory to LNT. C. Willis conducted K-40 deficient research at Oak Ridge National Laboratory, and his statement at the March 26, 1996 meeting of the U.S. Nuclear Regulatory Commission (NRC) clarified the situation (Muckerheide, 2000b).

6. DISCUSSION

If IR is infinitely harmful, as LNT would dictate, then a person hit 20,000 times each second would not be able to survive. This amount of IR is apparently indispensable to initiate basic biochemical reactions.
A major concern about IR is the induction of cancer. Human beings have excellent defense systems against IR, such as quenching ROS, repairing DNA damage, removing damaged cells by apoptosis, and killing most cancerous cells by immune systems. In fact, LNT is nothing but empty fear-mongering. Nevertheless, LNT advocated by NAS has been the basis of IR regulation until now (ICRP, 2007). It has hindered the rational development of nuclear power in medical, industrial, and other fields. One can readily cite several related points to promote medical development, hoping for the full development of radiological therapy.
Non-Hodgkin’s lymphoma can be improved by total or half-body X ray irradiation (Sakamoto, 2004). Radon inhalation therapy shows promise for cancers of four types: colon, uterine, lung, and liver cell (Kojima et al., 2019). Low dose radiation therapy (LDRT) is effective for treating Alzheimer’s disease (Cuttler et al., 2021). Of seven clinical trials, six have shown LDRT as successful for treating severely affected COVID-19 patients (Table 2 of Sutou, 2022). That effectiveness is apparently achieved by IR-induced macrophage phenotypes: M1 (immune-stimulatory and pro-inflammatory distress induced by >1 Gy and M2 (immune-suppressive and anti-inflammatory eustress by <1 Gy via the Nrf2 pathway (Calabrese et al., 2021).
Acknowledgments
The author’s great appreciation is extended to Scientists for Accurate Radiation Information (SARI) members who have provided useful information through mutual communications.

Health Benefits of Exposure to Low Dose Ionizing Radiation

ABSTRACT

Radiation effects on living systems have attracted researchers for more than a century. The radiobiological knowledge has considerably advanced over the years. Early studies on ionizing radiation action on the cells and organisms focused on the damage to cellular molecules and cellular structures. The deeper radiobiological studies on living systems revealed radiation induced direct and indirect damages to vital cellular molecules such as DNA, protein, membrane and others. It became established that cellular responses to high and low radiation dose exposures are different. High dose radiation doses (>1\text{ Gy}) cause deleterious biological damages resulting in mutations and eventually translated in acute radiation injuries often leading to cancer. On the other hand, effects of low doses of radiation (<100\text{ mSv}) to cells and organisms are found to stimulate signaling and DNA damage repair processes. Low dose effects are considered enigmatic and not-extrapolatable from high dose-linear effects. This chapter is devoted to elaborate the molecular mechanisms of low dose radiation exposure to living organisms and emphasize the need to change the perspectives from high dose associated carcinogenic risk to beneficial health effects taking clues from anti-inflammatory effects, gene activation and modification of immune responses. New radiobiological knowledge such as bystander effect, adaptive responses and cytokine augmentation are discussed in the context of carcinogenesis, low dose therapy, protection to health and novel COVID-19 treatment. It is suggested that the emphasis of scientific research of low dose exposures to be shifted from low dose exposure related cancer risks to possible beneficial effects on health e.g. radiation medicine to develop new diagnostic tools and technologies for human welfare.
Keywords: Low Dose Radiobiology; ROS; Radiation carcinogenic risk; Bystander Effect; Positive Health effects; Low Dose Radiation Damage Repair and Signaling; Low Dose Radioprotection; Low Dose Anti-inflammatory Effects and COVID

1. INTRODUCTION

Human population is constantly exposed to natural and man-made radiations but the adverse health effects of radiation became a topic of concern soon after the discovery of X-ray in 1895. Radiobiology studies pertaining to the biological effects of ionizing radiation in a wide range of doses set guidelines for addressing the safety issues of nuclear workforce, diagnostic and therapeutic medical professions. The knowledge of cellular and organism response to ionizing radiation allowed the development of many tools and methods for new applications to diagnosing and treating diseases. The field of medicine witnessed revolution and it became routine to diagnose many invisible diseases in the body, and internal organs and structures visualized using radiation based techniques. In medical radiography and cancer radiotherapy, techniques based on X-rays and computed tomography (CT) scans, gamma radiation, accelerators, permitted not only detection but also treatment of many diseases situated in the deep tissues in the human body that were otherwise impossible without surgical intervention. Radiobiology and radiation chemical studies revealed that ionizing radiation were able to break chemical bonds and ionize biomolecules resulting in damage to cellular molecules such as DNA. As research progressed, the DNA became known a central molecule of the cell and radiation damage to DNA was attributed to radiation cell death. Physicians were quick to apply this knowledge to kill cancer cells and saving life of millions of patients.
Apart from cancer treatment, physicians began heavy reliance on radiography to assess human health. With the increasing use of nuclear and radiation technologies in hospitals, the annual doses of radiation received by medical staff and general people showed rising trend. Further studies showed, that, unlike the carcinogenic effects of high dose exposure ascribed to DNA single and double breaks, the perceived risk from the low-dose radiation were considered insignificant or negligible as it does not cause any acute toxicity but suspicion continued for likely increase in the carcinogenic risk. Most often, the empirical linear fits of existing carcinogenic human data have been determined from high dose exposures, such as those of Japanese atomic bomb survivors (Preston et al. 1994). It was found that low-dose radiation exposures rarely induced DNA double strand breaks (DSB), which are considered the most relevant lesions for the deleterious effects of radiation (Rothkamm and Löbrich 2003). Notably, observations suggest that cumulative radiation doses of 50\text{ mGy} have long term detrimental health effects, including >3-fold increase in the risks of acute lymphoblastic leukemia and myelodysplastic syndrome (Pearce et al. 2012). But, more clinical studies are needed to validate the findings. Studies on mouse demonstrated that low-dose radiation affect the function of long-lived tissue-specific stem cells, including hematopoietic stem cells (Rodrigues-Moreira et al. 2017). Thus, understanding of effect on tissue-specific stem cells is of particular importance in evaluating the risks posed by low dose radiation.
The BEIR VII report provides a comprehensive description of health effects, both cancer and non-cancer diseases caused by the low dose radiation. This report defines low doses in the range of near zero up to about 100\text{ mSv} (0.1\text{ Sv}) of low-LET radiation. The units of radiation Gy and Sv are interchangeable for low LET radiation such as x ray and gamma ray. The BIER VII report of the National Academy of Sciences, USA published in 2006 provides an exhaustive cover on low dose radiation risk of cancer based on large body of epidemiological and radio-biological data. The Life Span Study (LSS) of the Japanese Atomic Bomb survivors, who received an acute dose of radiation, mostly in the form of \gamma-rays, with a small component of neutrons forms an important reference for human studies. In addition, studies of medically irradiated patients for diagnosis, imaging and occupational or environmental exposures constitute data base for low dose health effects. For thyroid and breast cancers, risk estimates are based on pooled analyses of the LSS and medically irradiated cohorts. While studies on populations exposed occupationally or environmentally have, so far, been of limited value in quantifying radiation risks, but they can provide valuable insight of cancer risks from chronic exposures. One of the raised issues in radiation risk assessment is how to extrapolate risk estimates derived from data on relatively high acute exposures in case of the LSS cohort to low doses or chronic exposure situations below about Thus, the question is how to extrapolate from an observed risk due to an instantaneous dose of 0.1\text{ Gy} or more to an extrapolated risk from a chronic low dose exposure. Although ionizing radiation is known to induce mutation in animal germ cells, but hereditary effects in humans have not been demonstrated. The genetic risks from low dose radiation exposures are often estimated mainly from cellular and animal studies. There are also research reports that radiation at moderate doses can induce non-cancer health effects, such as cataract and cardiovascular disease, and these effects may not have a threshold. However, unlike the case of radiogenic cancer and hereditary effects, there is, at present, neither direct evidence nor a strong theoretical basis for occurrence of such effects at lower or chronic radiation exposures.
Taking into account this background information and associated apparent limitations of basic understanding, this Chapter describes distinctions of high and low dose radiation effects, radiobiological mechanisms with relevance to diagnosis and therapy, low dose radiation related new phenomenon such as bystander (non-target) effects, and cellular and organisms adaptability and their relevance to linear-no-threshold issues and finally, an account of low dose new radiobiological results in beneficial and useful health applications.

2. HIGH AND LOW DOSE RADIATION EFFECTS AND CARCINOGENIC RISK

Radiation effects on cells and organisms are different at high doses (>1\text{ Gy}) and low doses (100\text{ mGy}) and dose rates. Effects of low and high doses of radiation was compared on hematopoietic stem and progenitor cells obtained from human umbilical cord blood. It was found that a single dose of 20\text{ mGy} radiation was sufficient to impair the self-renewing capacity of cord blood hematopoietic stem cells (Henry et al. 2020). Intriguingly, this effect is independent of canonical DNA damage response (DDR), as a 20\text{ mGy} dose fails to induce DSB markers \gamma\text{-H}_2\text{AX} and \text{53BP1} foci, or DDR hallmarks phospho-ATM and -p53, all of which are induced by a 2.5\text{ Gy} radiation dose. It is further found that low dose effect was mediated by reactive oxygen species (ROS) generated via the cell respiratory process of oxidative phosphorylation in mitochondria, and \text{p38/MAPK14} (Ito et al. 2006). Low-dose IR impair human cord blood hematopoietic stem cells function through ROS and \text{p38/MAPK14}, but not via canonical DDR via ATM or p53 (Henry et al. 2020).
Exposure of living organisms at high doses and high dose rate of ionizing radiation cause acute injuries leading to mutations and eventually cancer but low doses of radiation are limited by uncertainty of damage to cancer induction. Comparisons of radiation effects on gene activation at high and low doses show different responses although a small percentage of genes exhibit common responses. In 2006, the National Research Council of the National Academy of Sciences (NAS), USA released the BEIR VII report (National Academy of Sciences 2006) which reviewed recent evidence concerning the health risks from low-level, low linear energy transfer (LET) radiation. The BEIR VII Committee developed models for calculating the risks of radiogenic cancers, based on updated information on the A-bomb survivors, as well as other research data. In this report, the BEIR VII models employed methods to arrive at revised estimates of radiogenic risks for most cancer sites. BEIR VII risk estimates were derived for low doses of \gamma-rays with typical energies between about 0.1 and 10\text{ MeV}, with a brief discussion of possible enhancement of risk for more densely ionizing electrons and photons. Although the main focus in the subsequent report, as in BEIR VII, on low-LET risks, the evaluation of cancer risks to high-LET radiation (\alpha-particles) and an outline of a biophysical approach to estimate risks from low energy photons and electrons were discussed. Further report presented a quantitative uncertainty analysis which is based on different approaches than that in BEIR VII and which incorporates some additional sources of uncertainty. With reliable epidemiologic data at low doses, not available due to methodological limitations and confounding factors, future active biological researches are needed to supplement fundamental information on the mechanisms of low dose radiation effects.

3. RADIOBIOLOGICAL MECHANISMS

Mammalian cells produce ROS which are carcinogens, key actors of the non-specific immune defense against pathogens and, in a subtler way, signals of transduction, cellular metabolism. Oxidative stress can induce severe damage to the host which, in turn, adapted to face subsequent oxidative injuries. Disruption of redox balance leads to various pathological conditions, such as cancer. Therefore, studies are warranted on exploring the mechanisms linking ROS, cancer cells, anti-tumor immunity and therapy. It is important to understand the role of oxidative stress in tumor microenvironment and the correlation between ROS, proliferation and death of cancer cells. Interestingly, radiation induced granulocytes, as key inflammatory cells and ROS producers, are nowadays exploited for eradication of cancer cells. It is important to note that tumor cell redox balance is a suitable therapeutic target, either alone or in combination with other agents of cancer cells killing. It is presumed that the probability of carcinogenesis induced in an organism from an exposure to radiation is proportional to the number of induced mutations remaining after repair. This has led scientists the excess risk in mathematical modeling of dose-effect for a relatively high acute dose, with a reduction by a DDREF factor for low doses and dose rates. The DDREF for carcinogenesis would be equal to that for the underlying process of radiation-induced mutagenesis. Relatively recent findings point out that aerobic organism learned to take further advantage of apparently toxic ROS. Evidence exists that ROS are beneficially involved in many signaling pathways that control development and maintain cellular homeostasis. In physiological conditions, a tightly regulated redox balance protects cells from the injurious attack of ROS, but if altered, it promotes various pathological conditions. Understanding the duality of ROS as cytotoxic molecules and key mediators in signaling cascades, may provide novel opportunities for improved therapeutic intervention.

 CONCLUSIONS AND CHAPTER SUMMARY

Understanding of the low dose and dose rate effects on cells and organisms is fast growing but many of the observations are inconclusive and it merits greater research focus and resource allocation now than previously. The low-dose radiation emanates from diverse sources and different types of radiation e.g., alpha, beta, or gamma rays, routes of exposure (internal or external), and duration of exposure (acute or protracted). There is obvious gap of knowledge and there exists no direct link of low dose exposure and consequences to human health. Most often, low dose irradiation and their direct health effects, estimation of carcinogenic risks and other diseases are derived by using the same generic approach that relies on risk estimates obtained from higher, acute, external radiation exposures, largely of the Japanese Atomic Bomb survivors, and applying appropriate correction factors. In view of the new knowledge in radiobiological studies such as bystander effect, adaptive mechanism and individual radio-sensitivities, the concepts are changing on radio-sensitization for cancer radiotherapy and radioprotection for safety of occupational workforce and general people. More concerted and intensified radiation research are urgently warranted at cell, organism, organ, and laboratory animal levels to address scientific questions of radiation utility, regulation standards, modification in LNT model and, above all, health effects for developing newer diagnostic and therapeutic tools. New diagnostic tools such as PET, SPECT and radio-pharmaceuticals have become sought after procedures in nuclear medicine and reliable knowledge on safety and protection are not only necessary but urgent. In recent years, radiobiology and radiotherapy researches from animal and clinical studies have demonstrated that immune responses were stimulated by LDR, but the same were suppressed by high-dose exposures. More significantly, it has now been known that DNA repair is stimulated by low-dose exposures and is suppressed by high-dose exposures, making stronger case in favour of LDR therapies in the treatment of cancer and other diseases. However, more research is required to convince clinicians and develop this as one of the common modalities of cancer therapy.
It is most imperative to pool research resources and recruit talents to initiate coordinated multi-disciplinary low-dose radiation research program for national development and globally relevant improved understanding of adverse human health effects from exposures to radiation at low doses and dose rates for aiding in nuclear medicine clinic and radiotherapy departments. Obviously, vast scope exists to investigate mechanism in radiobiology for radiotherapy including radiobiology of cancer stem cells (CSCs). In deciding the vision for future research, more focus is needed on radiobiology-based research projects for deeper insights on low dose exposure effects (whole body or partial exposures) on health including cancer incidence as well as non-cancer diseases e.g. cardiovascular, cataract, nervous system. No compromise or delay is acceptable in efficiently discharging the scientific responsibilities to address human health issues by unravelling the secrets of biological organizations and functions and draw inspiration to develop technologies for future. Identifying the low radiation induced molecular mechanisms of DNA, membrane, cytoplasmic damages, signaling and repair mechanisms involved in directly irradiated and bystander cells/tissues would help open new vistas for radiobiological based applications. It is extremely interesting to unravel the underlying role of ROS in high and low dose radiation exposures at DNA damage and repair processes. It is demanded to develop improved risk models for doses and dose rates at which direct measurement of risks is not currently possible, and ultimately develop more individualized risk estimates for future medicine.
To develop a broader scientific understanding of low dose radiation health effects and satisfying the public anxieties of safety despite enjoying useful health benefits in diagnosis and therapies. It is highly warranted to support and engage young minds in radiation research to conduct novel research at cell, organism, and animal levels for generating seminal information adding value and seeking recognition among the international community.

Stem Cell Function and Tissue Regeneration in the Context of Exposure to Low Dose Ionizing Radiation
Ethan Karivelil¹ and Soji Sebastian

ABSTRACT

The health risks to humans exposed to low doses and low dose rates of natural, anthropogenic, occupational or diagnostic sources of ionizing radiation remain ambiguous. Whereas a large volume of laboratory and human epidemiological studies have shown that exposure to high doses of radiation engender significant health risks, accumulating experimental evidence suggests that the biological effects of low absorbed doses of radiation (LDR) are different from those of high doses. LDR stimulates proliferation of certain tissue specific stem cells for example mesenchymal stem cells and muscle stem cells and their regenerative properties. Such specificity is partly due to inherent radio-sensitivity rooted in the epigenome. Understanding the basis of these stimulatory responses is significant to translational applications in regenerative medicine. Here we provide an overview of the current literature on stimulatory radiation effects in stem cells and their potential clinical applications.
Keywords: Hematopoietic Stem Cells; Hormesis; Ionizing Radiation; Radiosensitivity; Mesenchymal Stem Cells.

1. INTRODUCTION

Decades of epidemiological and experimental research into the radiation effects on human biota has shown that a range of biological processes, ranging from molecular and cellular changes to physiological and mechanical tissue functions are affected. The nature of these changes, their magnitude and impact on health risks depend on the dose and dose rate received [1-3]. Health risks (mainly cancer) associated with human exposure to low dose ionizing radiation are currently estimated using the Linear-No-Threshold (LNT) model, meaning that any exposure will have adverse consequences. However, accumulating phenomenological and mechanistic advances in understanding the events associated with low dose radiation effects have revealed that exposure to LDR induces protective effects against spontaneously arising adverse effects and against damage from various environmental challenges. Thus the LNT model does not incorporate our current mechanistic knowledge of the biological pathways and possible multiple health outcomes, for example the regenerative capacity of tissues [4, 5]. Extensive evidence from epidemiological and experimental studies indicate that exposure to high dose radiation (HDR) results in DNA damage and subsequent chromosomal rearrangements leading to an increase in cancer rates, thus IR is considered a carcinogen, albeit a mild carcinogen compared to certain chemical agents [6].
A person receives an annualized average of 2.4\text{ mSv} from various environmental sources. The US National Academy of Sciences and the National Council on Radiation Protection and Measurements (NCRP), 2015. Health Effects of Low Doses of Radiation: Perspectives on Integrating Radiation Biology and Epidemiology. NCRP Commentary No. 24. Bethesda, MD: NCRP, have defined a low absorbed dose of low linear-energy transfer (LET) radiation to be a dose <100\text{ mGy} delivered acutely, and a low absorbed-dose rate is <5\text{ mGy h}^{-1} for any accumulated absorbed dose [7-9].
Interestingly, although there are habitable areas on the Earth with considerable population density, where natural background radiation differs by orders of magnitude higher than most other regions on the Earth (e.g., Ramsar in Iran, Kerala in India, Guarapari in Brazil), published reports suggest no increased health risk having been documented among the inhabitants [10]. In contrast, radiotherapy modalities deliver at higher orders of magnitude doses which results in collateral damage to the normal cells peripheral to the tumor upon exposure to gradients of radiation dose, therefore it is believed they also increase the chances to secondary cancers [11]. However, the association between LDR exposure and cancer incidences is controversial and lacks statistically significant epidemiological support. Moreover, numerous scientific reports have shown evidence for stimulatory and beneficial effects, collectively called radiation hormesis [12-16]. Radiation hormesis is a phenomina whereby low doses of radiation provoke stimulatory beneficial effects in normal cells. Epidemiological studies have shown that the risk of cancer induction following exposure doses up to 100\text{ mSv} (100\text{ mGy}) is uncertain due to limitations in size of the studied cohorts with certain studies suggesting no increase in risk [17]. Therefore, investigating the stimulatory effects of LDR on stem cells has potential benefits in tissue regeneration.
Most tissues in the body harbor adult tissue stem cells (SCs)-multipotent, quiescent, undifferentiated mono-nuclear cells, capable of self-renewal, proliferation and differentiation into mature functionally competent cellular subtypes [18, 19]. In the last decade, progress has been made in methods for isolation and moderate enrichment of these therapeutically valuable cells. However, due to number of limitations these efforts have yet to translate into efficient modalities to treat organ dysfunctions and failures. For example, one of the limitations is related to low yields of stem cells not sufficient for therapeutic transplantation purposes [20]. Ex vivo expansion may improve the final yield; however, rounds of passaging typically lead to loss of stem cell characteristics, accumulation of DNA damage emanating from telomere shortening, higher immunogenicity and other undesirable outcomes [21]. Subsequently, these can result in immunological rejection post transplantation and inefficient tissue formation. In addition, such transplanted progenitor cells are unable to travel great distances from the point of injection, causing poor integration into host tissues. It is not surprising that clinical trials using adult stem cells for treating organ dysfunction have not yet been successful. Alternative approaches include the use of pluripotent human embryonic stem cells (hES) or induced human pluripotent stem cells (ihPS). Yet, lengthy protocols of directed progenitor cell generation from hES or lhPS cells may still suffer from issues in general related to in vitro growth and expansion and in particular somatic mutations [22-24]. Thus, there is a need for pre-conditioning of ex vivo maintained cells that would preserve/improve the stem cell identity and other qualities that are key to successful therapy.

2. HEALTH EFFECTS OF LDR ON HEMATOPOIETIC STEM CELLS (HSCs)

Decades of research surrounding stem cells, has shown that hematopoietic stem cells (HSCs), which reside in the bone marrow hypoxic condition are highly radiosensitive [25]. The HSC system is an essential organ system, due to its primary function of generating mature functional cells that circulate in the blood and participate in immune and other functions, known as hematopoiesis. Notably, a single HSC has the potential to re-populate the entire blood system, and henceforth, HSCs are classified as Long Term Repopulating Cells (LT-HSCs) and Short Term Repopulating Cells (ST-HSCs) based on their reconstitution ability [26]. LT HSCs (\text{CD34}^-\text{ Flk2}^-\text{ Lin}^-\text{ c-Kit}^+\text{ Sca-1}^+) are a population of quiescent cells residing in the bone marrow (BM) with a self-renewal capacity retained for greater than 6 month. However, \text{ST-HSCs} (\text{CD34}^+\text{ Flk2}^-\text{ Lin}^-\text{ c-Kit}^+\text{ Sca-1}^+) are lineage – committed, depending on the niche’s intrinsic and extrinsic signals, and thus cannot sustain their self-renewal property for more than a month [27]. ST-HSCs differentiate into hematopoietic progenitor cells (HPCs), which further segregate into common myeloid progenitors (CMPs) and common lymphoid progenitors (Fig 1).
Bone marrow (BM), peripheral blood (PB) and cord blood (CB) are the proven source of HSCs for therapeutic applications and biological studies. Collective analysis of HSCs in PB is useful due to its availability and accessibility [28]. However, the prospective isolation and subsequent expansion of HSCs is difficult due to inherent heterogeneity of the HSCs regardless of its origin; consequently generation of sufficient HSCs with long term repopulation potential for better engraftment needs pretreatment without compromising its stem cell features. Exposure to high dose radiation elicits adaptive response in HSCs, in consistent with a strong activation of p53-mediated DNA damage and DNA repair through Non-Homologous End Joining (NHEJ) that leads to genomic instability [29] [30].
It has been widely believed that, exposure to LDR may not prompt severe responses in HSCs. However, detailed analysis indicative of long term defects in hematopoietic stem cell functions due to hyper radio sensitivity; i.e. an observed defect in exposed HSCs with regard to long term repopulating ability in functional hematopoietic assays. Here, we surveyed the literature on the biological effects of radiation hormesis in HSCs exposed to LDR as a potential pretreatment for stem cells and regenerative medicine. Doses below 100\text{ mGy} in LT-HSCs is indicative of hyper-radio sensitivity, specifically 20\text{ mGy} single acute dose is not associated with an early DNA damage and apoptosis. Gene expression analysis of 20\text{ mGy} irradiated HSCs showed that the early response to radiation is independent of p53 pathway and specifically regulated a set of genes linked to Keap1/Nrf2 pathway, involved in regulating oxidative stress via Reactive Oxygen Species (ROS), mitochondria function and radio-resistance [31]. The long term effects of a 20\text{ mGy} exposure caused persistent oxidative stress as a result of an impaired mitochondrial function, defective stem cell self-renewal, thus premature exhaustion of the LT-HSCs. Consistent with 20\text{ mGy }\gamma, a similar exposure (20\text{ mGy}) from an X-ray source induced ROS generation, consequently; the exposure did not elicit DNA damage or apoptosis, rather inhibited the self-renewing capacity of CB derived HSCs, alluding to p53 independent effects similar to \text{LDR }\gamma exposures [32].

3. EFFECTS OF LOW DOSE RADIATION ON PERIPHERAL BLOOD MONONUCLEAR CELLS (MNCs)

Perhaps the best living evidences for radiation hormesis are from studies conducted in the High Level Natural Background Area (HLNRA) of Kerala coast (with radiation dose extending from <1.0 \ge 45\text{ mGy}). This geographic region is very unique due to chronic exposure to radiation emitted by thorium and its decay in its monazite rich sand [34]. Epidemiological studies carried to survey for adult cancer incidents, congenital abnormalities, heart diseases and mental disabilities did not find statistically significant adverse effects [35-39]. On a mechanistic level, compared to inhabitants in control region, measurement of telomere length, various forms of DNA damage, including micronuclei, did not find substantial changes [37]. However, cellular studies using peripheral blood mononuclear cells (MNCs) using formation of \gamma\text{H2AX} foci and micronuclei formation found a better DNA repair efficiency exist in MNCs of elder/aged individuals residing in HLNRA (>5.0\text{ mGy/year}) when these MNCs were challenged with higher \gamma ray doses. These results are indicative of expression of adaptive responses in the HLNRA inhabitants [40]. A detailed omic analysis by a combination of gene expression and proteomics in persons residing in the HLNRA region uncovered dose dependent responses in the expression levels of genes/proteins involved in diverse biological process such as DNA repair, cell cycle checkpoint regulation and chromatin remodeling, suggestive of induction of protective mechanisms against harmful effects [41] [42, 43]. As DNA repair and associated chromatin remodeling positively regulates stem cell functions in aged individuals, this may lead to an improvement in tissue regeneration in elderly individuals, within which the long term regenerative potential of hematopoietic compartment is directly dependent on the LT-HSCs; thus there is a need to investigate the biological hormetic effects exclusively on LT-HSCs from MNCs. Single cell sorting followed with single cell RNAseq (scRNAseq) can be an effective strategy to uncover the influence of radiation hormesis on cell type specific lineage progression and hematopoietic homeostasis. Consequently, the regenerative potential is directly linked to the epigenetic memory within which loci specific histone 3. 3 modifications, in tandem with DNA methylation; forms the basis of HSCs lineage progression and youthful supply of progenitor cells for lifetime hematopoietic blood cell functions [44, 45].

4. APPLICATIONS OF LDR EXPOSED MESENCHYMAL STEM CELLS

Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent stem cells with the capacity to differentiate into mesenchymal lineage such as osteocytes, adipocytes, chondrocytes and myocytes. MSCs express cell surface markers such as CD29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34 and CD45 [46]. MSCs for the first time were reported in the BM [47], and now they have been isolated from various tissues for example CB, PB and dental tissues, adipose tissues and endometrium. MSCs are known for their in vitro proliferative expansion without compromising lineage potential and differentiation commitment. Cell based therapy using MSCs is a desired option to treat acute tissue damage and chronic fibrogenesis. MSCs administration could repair injured lung, liver and heart by reducing inflammation owing to their immunomodulatory properties. Thus, MSCs are an attractive cell source for regenerative medicine as reviewed in Pittenger et al. 2019 [48]( Fig 2).

5. REGENERATIVE POTENTIAL OF MUSCLE STEM CELLS EXPOSED TO LDR

Skeletal muscle is one of the largest organs in the human body and its long term maintenance depends on muscle stem cells (MuSCs). The latter represent the major population of the resident stem cells in adult skeletal muscle. These adult stem cells facilitate the postnatal growth, remodeling and repair of the muscle tissue. Upon muscle injury, routinely experienced by humans during physical activity, MuSCs relocate by traveling great distances from the surface of the muscle fibers to the area of injury across the muscle mass and proliferate extensively [53, 54]. During the process, MuSCs adopt one of two fates: (1) self-renewal, a process to replenish the existing pool of quiescent stem cells; (2) differentiation to form myoblasts that will fuse with damaged muscle fibers to repair the muscle (Fig. 3). In fact, the remarkable regenerative capacity of skeletal muscle made it one of the best studied organs in mammalian tissue regeneration [55]. Consequently, there is a great promise for treatment of muscle diseases using MuSCs. Muscle diseases are broadly classified as (1) genetic (e.g., Duchenne Muscular Dystrophy), (2) aging related sarcopenia, (3) muscle wasting cachexia, and (4) type 2 diabetic myopathy [56].
In the last decade, progress has been made in methods for prospective isolation and limited propagation of MuSCs [57]. However, due to a number of limitations, these efforts have yet to translate into efficient modalities to treat muscle diseases. For example, one of the limitations is related to low yields of MuSCs that are not sufficient for therapeutic transplantation purposes. Ex vivo expansion may improve the final yield; however, rounds of passaging typically lead to loss of proliferative expansion, accumulation of DNA damage, precocious differentiation and senescence. Subsequently, the aforementioned reasons cumulatively result in immune rejection post transplantation and inefficient repair of the damaged muscle tissue [58]. Further, the transplanted cells are unable to travel greater distances from the point of injection, causing poor integration into host tissues. It is not surprising that clinical trials using MuSCs for treating muscle diseases have not been successful. Alternative approaches include the use of pluripotent human embryonic stem cells (HESCs) or induced human pluripotent stem cells (iHPS). Yet lengthy protocols of directed muscle stem cell production from HES and iHPS cells may still suffer from issues related to in vitro growth and expansion issues. Therefore, there is a need for preconditioning of in vitro or ex vivo maintained cells that would preserve/improve their muscle and stem cell identity and other qualities that are key to a successful therapy.

6. CONCLUSION

Although hormesis has been manifested under certain experimental conditions, challenges in how to translate the mechanism from a well-controlled in vitro heterogeneous stem cell models to clinical settings need to be addressed. In general, LDR responses are highly cell type specific with a remarkable degree of variability both between individuals and different cell types owing to the inherent radiation sensitivity, revealed in gene expression studies and potentially rooted in stem cell specific epigenetic memory. To promote the clinical application of LDR hormesis, appropriate humanized mouse models that mimic human response to LDR would be useful to unravel the mechanisms underlying potential regenerative effects.
Acknowledgements
Authors are thankful to Prof. Edouard Azzam and Ms. Candice Didychuk for their help with revisions. Authors would like to thank Federal Science and Technology (FNST) program, Canada for the financial support.