Radioprotective-A pharmacological intervention for protection against ionizing radiations: A review
Y Joshi, T Jadhav, V Kadam
Keywords
antioxidants, immunomodulators, radioprotectives, thiols
Citation
Y Joshi, T Jadhav, V Kadam. Radioprotective-A pharmacological intervention for protection against ionizing radiations: A review. The Internet Journal of Internal Medicine. 2009 Volume 8 Number 2.
Abstract
When harmful ionizing radiations are exposed to humans, harmful effects and even death may occur. In 1940’s it was proved that endogenous thiols like cysteine showed protection against such harmful radiations. Later, various synthetic drugs containing thiols were developed and proved to be highly effective. But due to their toxicity, the research and development is more inclined towards the discovery of herbal medicines and other naturally occurring antioxidants as radioprotectives. The recent trend is the use of immunomodulators to regenerate the radiation injury by development, proliferation & differentiation of the hematopoietic stem cells and progenitors.
Introduction
Ionizing radiations produces deleterious effects in the living organisms. The rapid technological advancement has increased human exposure to ionizing radiations enormously. Widespread use of radiation in diagnosis therapy, industry, energy sector and inadvertent exposure during air & space travel, nuclear accidents & nuclear terror attacks requires safeguard against human exposures. Lead shielding and other physical measures can be used in such situations but with difficulty to manage; thus pharmacological intervention could be the most prudent strategy to protect humans against the harmful effect of ionizing radiations. These pharmacological agents are radioprotectives.
Radioprotective effects for a bacteriophage were observed by Latarjet and Ephrati in 1948, using cysteine, cystine, glutathione, thioglycolic acid, and tryptophan. The use of the mercapto/thiol functions was demonstrated in 1951 by Bacq, a Belgian physiologist, who removed the carboxyl group of cysteine & obtained 2-mercaptoethylamine NHCH2CH2SH(MEA, or cysteine amine), which proved to be a much stronger protective agent in mice than any previously tested.
The discovery of MEA was a stepping stone in the development of various other radioprotectives.
Most of the mercaptans and other thiol molecules, later synthesized, also contained an amino or other basic function showing the activity. MEA and its derivatives, particularly those having greater lipophilicity are most potent one.
In recent years, radioprotective agents with a novel mode of action have been investigated; in particular, compounds that can affect haematopoietic stem cell regeneration have attracted significant interest. The aim of this strategy is to increase survival rate by stimulating the function and regeneration of the stem cell population that is decreased, due to radiation induced damage. Immunomodulators and cytokines represent the bulk of agents in this category. Naturally occurring compounds that function as antioxidants and immunostimulants are another strategy for the development of radioprotective agents with low toxicity. Therapeutic agents that can be administered following irradiation are another strategy for reducing side effects induced by ionizing radiation; cytokines and immunomodulators, through induction of bone marrow recovery and extrahaematological tissue regeneration can represent such a class of agent.
Radiation Injury
Ionizing radiation
Ionizing radiation are electromagnetic radiation (such as X or gamma rays) or particulate radiation (such as neutrons or alpha particles) that has sufficient energy to ionize atoms or molecules; that is, to eject electrons from their outer orbits.
In considering the effects of radiation on biological systems, it is important to distinguish the different types of ionizing radiation according to their LET (low energy transfer). LET is the amount of energy deposited by a particular type of radiation per unit of path length. Low-LET radiation (X and gamma rays) is sparsely ionizing because it causes little ionization per micron of path length, whereas high-LET radiation (neutrons and alpha particles) is densely ionizing because it produces much ionization per micron of path length. Generally, high-LET radiation is much more efficient in producing biological damage than low-LET radiation.
Biological Damage
Death from radiation injury is the result of a sequence of events that occur over a period of less than a billionth of a second to several weeks. The first step in this sequence is the transfer of radiation energy from the photon or particle to atoms and molecules in its path. This results in a chemical alteration in macromolecules that are critical for biological function. The importance of membrane damage is still being evaluated; much of the evidence suggests that damage to DNA.
The injury to the biological matter by radiation takes place in two ways:-
Direct action- It involves absorption of radiation energy by target molecules, such as DNA or RNA, resulting in molecular damage.
Indirect action- If the molecule is not in the radiation path, it can still become chemically altered indirectly, via reactions with free radicals and reactive oxygen species produced primarily from the radiolysis of water.
The radiolysis of water is,
Lifetime of the radiolysis product of water in solution is sufficiently long to allow them to diffuse and extend the damage beyond the primary path of radiation hence the effects of ionizing radiation within the cell are greatly amplified. The net effect of direct and indirect damage is the disruption of molecular structure and function, leading to altered cell metabolism. When DNA is damaged, this is followed by altered cell division, cell death, depletion of stem-cell pools, organ system dysfunction, and, if the radiation dose is high enough, death of the organism.
Lipid per oxidation takes place after irradiation or free-radical attack. This leads to the production of short chain fatty acyl derivatives, lipid-lipid cross-linking as well as protein-protein and lipid-protein cross-linking, oxidation of accessible amino acids, protein denaturation, and scission of disulphide bonds in proteins. Functionally, these changes can be expressed as altered membrane fluidity and permeability, which could trigger the release of potent physiological mediators. Activity of enzymes associated with these membranes may be altered by the disruption of lipid microenvironment and protein structure.
Protection against radiation injuries
Almost nothing can be done pharmacologically to protect against the initial transfer of radiation energy to either water or critical biological molecules as the transfer occurs too rapidly (within 10-14 seconds after irradiation) and is a purely physical process. That is radioprotective agents fail to protect against direct damage to critical molecules. Protective agents would be most effective against a low-LET radiation hazard. Protection depends on the ability of chemical agents to reduce the intracellular concentration of free radicals and reactive oxygen species that are produced within the first millisecond after irradiation.
Mechanism
The damage induced by the products of radiation interactions with water can be reduced either by inhibiting the formation of these reactive radical intermediates, or by eliminating them from the cellular environment.
Free-radical scavenging
Free-radical scavenging is the ability of chemicals and endogenous enzymes to remove products of water radiolysis and highly reactive oxygen species before they can damage molecules of biological importance. These are competitive reactions between protective agents and biological molecules. In aqueous solutions, protective agents and enzymes react with free radicals and oxygen species to form relatively stable, nontoxic end products, thereby reducing the concentration of these reactive species and sparing the biological target. Many protectants are very efficient scavengers of water-derived free radicals.
Hypoxia
The extent of radiation damage in a tissue is directly related to the degree of oxygenation of that tissue; agents capable of reducing oxy-genation will mitigate the injury. Many of these chemical agents are known to induce transient systemic or localized hypoxia. Systemic hypoxia can be achieved in several ways: induction of hemodynamic cardiovascular alterations, interference with hemoglobin function, increased tissue oxygen utilization, and depressed respiratory-center function. At the cellular and molecular levels, localized hypoxia can be achieved by agents that take part in the chemical and biochemical reactions that use oxygen. The usefulness of this mechanism must be considered with caution because of the potential effects of hypoxia on normal physiological function. This caution may apply more to agents that induce a systemic hypoxic state than to those that create localized hypoxia.
Mixed disulphide formation
The hypothesis was proposed by Eldjarn and Pihl. radioprotective thiols form mixed disulfides with thiol groups of proteins. The mixed disulfides provide protection to the protein thiols by interfering with indirect radiation damage from radiolysis products of water.
The mixed disulfide bond would be cleaved by radical scavenging and thus protecting the protein.
Non-protein sulphydryl release
Release of endogenous thiols (GSH, l-cysteine, or N-acetyl-l-cysteine) is one of the metabolic effects of the radioprotective thiols. This increase cellular thiol content is often 30 to 40 fold greater than the amount of thiol supplied by the protective agent. In some studies done by scientist, the natural radioprotectivity of mice was related to the concentration of thiol groups in the blood-forming tissues of the spleen. Development of radio resistance in cells was attributed to increased concentration of non-protein-bound thiols.
Control of DNA breaking
Ability of the disulfides of the radioprotective amino thiols to bind reversibly to DNA, RNA, and nucleoproteins has been postulated as a result of in vitro studies. This, according to Brown, can result in two restorative effects: first, the loose ends of the helix resulting from single-strand rupture are held in place, so that shortening or alteration of the chain is prevented; and second, the replication rate of DNA is decreased or halted, so that repair can take place before radiation-induced alterations are replicated. This binding, together with either radical scavenging or repair by proton donation, provides a possible route of protection of the nucleic acids by the amino thiols.
Other strongly protective derivatives of MEA and MEG, such as the thiosulfate, phosphorothioate, trithiocarbonate, or acylthioesters, readily undergo disulfide formation.
Hydrogen transfer
Radiation damage to a critical biological molecule results in the transformation of that molecule into an organic free radical. In this form, the molecule can then react with oxygen or other free radicals and become permanently altered chemically. However, if a suitable hydrogen donor is in the vicinity of the damaged molecule, it can compensate for the damage by donating or transferring a hydrogen atom. Hydrogen atom transfer can be thought of as an instantaneous repair process, in which the original molecular structure is restored before the damaged critical molecule becomes permanently altered by further chemical reaction. Many of the agents that function as free-radical scavengers also have the ability to donate a hydrogen atom.
No radioprotective agents that function primarily or exclusively by chemical repair are available. However, the aminothiols, which act as free-radical scavengers, are all capable of hydrogen transfer and therefore can function in the repair strategy.
Oxygen detoxification
It is brought about by enzymes like super oxide dimutase. Although cells and tissues are equipped with endogenous enzymes (e.g. superoxide dismutase) capable of the detoxification and removal of the products of water radiolysis, when these reactive oxygen species increase in the biological system following exposure to irradiation, the endogenous system is incapable of protecting cells from the hazardous effects of free radicals.
Superoxide dismutase (SOD) enzymes are naturally occurring intracellular enzymes which scavenge O2 - by catalyzing its conversion to hydrogen peroxide and oxygen. The copper, zinc and manganese containing SOD (Cu, Zn, Mn and SOD) mimic the activity of the endogenous SOD. A pharmaceutical version of a copper–zinc-containing SOD has been marketed under the name of Orgotein, which has been used for ameliorating radiation side effects in patients.
Also, manganese complexes of kojic acid and 7-hydroxy flavone showed potent SOD activity and lipid peroxidation inhibitory activities in vitro. The SOD activity of the Mn complex of kojic acid was 10,000 times as potent as kojic acid itself.
Kojic acid (C6H6O4; 5-hydroxy-2-(hydroxymethyl)-4-pyrone) is a chelation agent produced by several species of fungi, especially Aspergillus oryzae.
It has been shown that manganese(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+, MnP) and other derivatives of SOD proved to be radioprotective.
Regeneration after radiation injury
Here the aim is to increase survival by stimulating the function and regeneration of stem-cell populations that have decreased in number due to radiation-induced cell death. Conceptually, this strategy can be applied to any organ system (such as the hematopoietic system and the gastrointestinal system) that relies on stem-cell proliferation to provide mature differentiated cells for proper functioning. However, because hematopoietic stem cells are the most radiosensitive, only regeneration of the hematopoietic system is now taken into consideration.
Immunomodulators
Agents like immunomodulators are able to induce haematopoietic cytokines. Immunomodulators are noncytokine drugs that have been proposed as an alternative to stimulate haematopoietic stem cells. The release of cytokines through the effect of immunomodulators can stimulate growth, differentiation and proliferation of haematopoietic progenitor and stem cells. In this way, this agent may protect and repair through enhanced production of bone marrow cells, circulating granulocytes, lymphocytes and platelets.
Cytokines
Ionizing radiation affects haematopoietic tissues and reduces the neutrophil and platelet numbers. Reduction in these circulating blood cells can result in septicaemia, haemorrhage, anaemia and death. One of the strategies for novel radioprotective agents is the stimulation, maintenance and proliferation of progenitor cells from bone marrow. Cytokines can stimulate haematopoietic stem cells. Administration of IL-1 (100 ng), IL-6 (200 ng) or IL-1 + IL-6 to mice 20 hours before exposure to 9.5 Gy resulted in survival rates of 76.9, 71.4 and 84.6%, respectively, compared to 13.3% in control animals. In some studies done by researchers combination protocols with different cytokines have been shown to enhance neutrophils and platelet recovery after irradiation and enhance the survival rate in irradiated animals.
Herbs & natural antioxidants
Natural compounds in the diet provide functional antioxidants. Reduction of oxidation damage by such natural antioxidants provides a degree of protection against ionizing radiation injury. Vitamin E (alpha tocopherol) & Vitamin C and related analogues are nutraceuticals that can scavenge singlet oxygen and superoxide- anion radicals. Vitamin E, administrated at a dose of 400 IU/ kg s.c. before irradiation in mice, showed an increase in survival rate of up to 79% versus 4% in the vehicle-treated control group.
Melatonin (N-acetyl-5-methoxy tryptamine) is a hormone produced by the pineal gland that scavenges hydroxyl and peroxyl radicals and peroxynitrite anions. Administration of a single oral dose (300 mg) of melatonin to healthy human volunteers reduced the frequency of chromosomal aberrations and micronuclei induced by irradiation in cultured lymphocytes.Melatonin is found in highest concentrations in cell nuclei, which contain the most sensitive targetmolecule for ionizing radiation, namely DNA.
Flavonoids are a family of polyphenolic compounds found in fruits and vegetables; as such, flavonoids exhibit strong antioxidant activities. In some studies, flavonoids significantly protected mice bone marrow cells, when administrated at low doses before exposure to radiation. The protective effect of flavonoids in mice may be attributed to their direct hydroxyl radical scavenging potency and thus behave like an endogenous enzyme.
The proposed radioprotective efficacy of plant extracts is as a result of their containing a large number of active constituents, such as antioxidants, immunostimulants and compounds with antimicrobial activity.
Short-term in vitro tests can provide a basis for detailed evaluation of radioprotective activity. The simplest tests could be the evaluation of lipid peroxidation in vitro. Assay of free radicals and antioxidant status of a pharmacological agent can also provide some leads regarding the radioprotective potential of such agents. If a plant or a natural product is found to inhibit lipid peroxidation and scavenge free radicals, it may act as a possible radioprotector. The next step is to evaluate its radioprotective potential in vitro using cell survival and micronuclei assays. If it is found to elevate cell survival and reduce radiation-induced micronuclei formation, it certainly has a potential as a radioprotector.
Citrus extract, Hawthrown fruit extract, abana extract, extract of Amaranthus paniculatus Ginger, Liv-52 methanolic extract of Grewia asiatica (Phalsa) fruit, triphala, Curcuma longa-Curcumin (Di-feruloyl-methane) and curcuminoids isolated, extracts of Terminalia arjuna, Terminalia chebula & Wagatia spicata also show radioprotective activity. Dehydrozingerone (DZ) is an important constituent of ginger (Zingerber officialanis) also showed the activity.
Using the micronuclei assay it was proved that grapefruit flavanone naringin showed the radioprotective activity. Also, hesperidin (HES) a flavonone glycoside, belonging to the flavonoid family found in citrus species showed the activity using the micronuclei assay.
Both orientin and vicenin obtained from the extract of Ocimum sanctum protect against death from gastrointestinal syndrome as well as from bone marrow syndrome when injected intraperitonially before whole body exposure to lethal y-radiation.
Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavone, present in Terminalia arjuna Scutellaria rivularis, Scutellariae radix and Scutelleria baicalensis has antioxidant effects thus also proved as a radioprotective.
Aqueous extract of fresh nutmeg mace (aril of the fruit of Myristica fragrans) were shown to possess antioxidant properties and showed radioprotective activity in mammalian splenocytes.
Naturally occurring polyamines like spermine and spermidine protect the DNA against radiation-induced single- and double-strand breaks.
The radioprotective properties of polyphenols from Gentiana dinarica Beck, family Gentianaceae were evaluated for their radioprotective activity in cultured human peripheral blood lymphocytes exposed to 2 Gy of gamma radiation in vitro. They successfully showed the activity.
The majority of plants and herbs contain polyphenols, scavenging of radiation-induced free radicals and elevation of cellular antioxidants by plants and herbs in irradiated systems could be leading mechanisms for radioprotection. The polyphenols present in the plants and herbs may upregulate mRNAs of antioxidant enzymes such as catalase, glutathione transferase, glutathione peroxidase, superoxide dismutase and thus may counteract the oxidative stress-induced by ionizing radiations. Upregulation of DNA repair genes may also protect against radiation-induced damage by bringing error free repair of DNA damage. Reduction in lipid peroxidation and elevation in non-protein sulphydryl groups may also contribute to some extent to their radioprotective activity.
Combination Agent
It is impossible for any protective or repair agent either to completely eliminate all of the reactive intermediates formed or to repair all of the damaged molecules. Regardless of the efficiency of scavengers and repair agents and their concentration within the cell at the time of irradiation, some molecular damage and cell death will occur. Agents that function in the three strategies (protection, repair, and regeneration) contribute in different ways to the mitigation of radiation injury. Effectiveness of agents that function in the regeneration strategy is limited because the agents require a pool of surviving functional cells on which to work. That pool of highly radiosensitive hematopoietic stem cells becomes depleted after fairly low radiation doses.
Ideal Radioprotective Drug
The ideal radioprotective agent should fulfill several criteria:
(a) It must provide significant protection against the effects of radiation.
(b) It must have a general protective effect on the majority of organs.
(c) It must have an acceptable route of administration (preferably oral, or alternatively intramuscular).
(d) It must have an acceptable toxicity profile and protective time-window effect.
(e) It must have an acceptable stability profile (both of bulk active product and formulated compound).
(f) Have compatibility with the wide range of other drugs that will be available to patients or personnel.
Unfortunately, to date, there is no radioprotector that fulfills all these criteria.
Limitations of the radioprotective drugs
(a) These have to be administered before irradiation.
(b) Their toxicity cannot be totally eliminated.
(c) These are not always suitable for oral administration.
(d) The metabolic products take long time for excretion.
Toxicity
Acute side effects (such as nausea, vomiting, and hypotension) are common, especially with the sulfur compounds.
Deliverability
Oral administration is the most desirable route, but this may be difficult to achieve. Transdermal administration (for example, via a dermal patch) is also acceptable, but is limited by the fact that only microgram or smaller quantities of the drug can be delivered. Most of the agents under study are effective in milligram to gram quantities. The major exceptions are the cytokines. The transdermal route may have greater applicability. The next most acceptable route of administration is sublingual. The least acceptable practical method is intramuscular injection. Intravenous and subcutaneous injection and suppository administration are unacceptable routes for a self-administered field-deployed drug. If taken as a liquid, either orally or parenterally, the agent must also be soluble and stable in a vehicle that is appropriate for administration.
Evaluation
The magnitude of chemical protection against radiation damage is most commonly assessed either by comparing percentage survival between the treated and the control groups at a selected lethal radiation dose, or by computing a dose reduction factor (DRF) for the drug under study. Percentage survival requires fewer animals and is more easily determined than DRF.
Amifostine (WR2721)
2-(3-aminopropylamino)ethylsulfanyl phosphonic acid
Amifostine is the radioprotective adjuvant used in cancer chemotherapy that has been clinically approved by the Food and Drug Administration (FDA) for mitigating side effects (xerostomia) in patients undergoing radiotherapy. It has been proved by in-vitro & in-vivo studies that it acts as a radioprotective. It is marketed by MedImmune under the trade name Ethyol.
Common side effects of amifostine include hypocalcemia, diarrhea, nausea, vomiting, sneezing, somnolence, and hiccoughs.
MOA- Amifostine is an inactive prodrug that cannot protect until dephosphorylated to the active metabolite, WR-1065, by alkaline phosphatase in the plasma membrane, scavenges free radicals.
Amifostine has been investigated for its effect in patients with head and neck squamous sell sarcinoma. It has also showed its effectiveness in acute & chronic esophageal injury in rodents. Also, the radioprotective effects of subcutaneously (SC) administered amifostine have been investigated in animal studies.
Radioprotection in patients with prostate cancer clinical trial of endorectal amifostine was also successfully investigated.
Other organosulfur containing agents like the naturally occurring presents in Allium sativum, plants of the onion species and cruciferous vegetables are important among the sulfur-containing plants ahowed the effect. Garlic and its organosulfur compounds like diallyl sulfide (DAS), diallyl disulfide (DADS), ajoene, allicin, allyl mercaptans and allyl methyl sulfides also proved to be radioprotective.
Conclusion
The development of radioprotective agents is important for protecting patients from the side effects of radiotherapy, as well as the public from unwanted irradiation.
The development of radioprotective agents has been dominated by the study of sulfhydryl compounds, particularly the aminothiols.
However, design and identification of new chemicals with low systemic toxicity and DRF values of 1.5 or more still holds promise for the future. Natural products
(herbal preparations) from plants, fruits, leaves,etc, has received much attention in the last decade and appears to be favourable in many respects as compared to chemical radioprotectors. These include lower toxicity in human beings (as many of these are used in alternative medicine in Asian countries for centuries), are easily available and in expensive, and have shown good radioprotection in preclinical studies.
If the synthetically prepared radioprotectors are so designed that these will give minimum toxicity and maximum efficacy, these may also be developed further.
More recent studies focused on developing drugs with moderate efficacy, low toxicity and that can be administered easily. Today, there are two strategies to find an approved radioprotective agent: immunomodulators and natural herbal medicine. Although cytokines directly act on the production and proliferation of hematopoietic system, they have limitations, such as inflammatory response and unfavourable administration route.
A new approach is to find a safe chemical or biological compound capable of binding cytokine receptors and hence, stimulating the release of two or more cytokines and indirectly regenerating haematopoietic stem cells. Another strategy is to find natural products with less toxicity & high efficacy.