Radiation-induced Free Radicals Research Articles

Electron paramagnetic resonance spectroscopy in radiation research: Current status and perspectives

Exposure to radiation leads to a number of health-related malfunctions. Ionizing radiation is more harmful than non-ionizing radiation, as it causes both direct and indirect effects. Irradiation with ionizing radiation results in free radical-induced oxidative stress. Free radical-mediated oxidative stress has been implicated in a plethora of diseased states, including cancer, arthritis, aging, Parkinson's disease, and so on. Electron Paramagnetic Resonance (EPR) spectroscopy has various applications to measure free radicals, in radiation research. Free radicals disintegrate immediately in aqueous environment. Free radicals can be detected indirectly by the EPR spin trapping technique in which these forms stabilize the radical adduct and produce characteristic EPR spectra for specific radicals. Ionizing radiation-induced free radicals in calcified tissues, for example, teeth, bone, and fingernail, can be detected directly by EPR spectroscopy, due to their extended stability. Various applications of EPR in radiation research studies are discussed in this review.

Viscosity changes in hyaluronic acid: Irradiation and rheological studies

Hyaluronic acid (HA) is a significant component of the extracellular matrix (ECM), particular interest being shown herein in synovial fluid. The present study aims to investigate the degrading effects of X-ray radiation on HA at radiotherapy doses. Measurements of viscosity and shear stresses on HA solutions have been made at different shear rates using various types of viscometer for different concentrations in the range 0.01-1% w/v of HA. The HA has been subjected to doses of 6 MV photon radiation ranging from 0 to 20 Gy, the major emphasis being on doses below 5 Gy. It is found that there is a dose-dependent relationship between viscosity and shear rate, viscosity reducing with radiation dose, this being related to polymer scissions via the action of radiation-induced free radicals. The dependency appears to become weaker at higher concentrations, possibly due to the contribution to viscosity from polymer entanglement becoming dominant over that from mean molecular weight. Present results, for HA solutions in the concentration range 0.01% to 1% w/v, show reduced viscosity with dose over the range 0-4 Gy, the latter covering the dose regime of interest in fractionated radiotherapy. The work also shows agreement with previous Raman microspectrometry findings by others, the possible bond alterations being defined by comparison with available published data.

Retinal phosphenes and discrete dark noises in rods: A new biophysical framework

Spontaneous rhodopsin activation produces discrete noises indistinguishable from single-photon responses. However, there is a serious discrepancy between the apparent energy barrier of thermal events compared with that of the photon-driven process. Current estimates of the activation energies of discrete dark noises in vertebrate rod and cone pigments are ∼40–50 kcal/mol for activation by photon and ∼20–25 kcal/mol for activation by heat. To reconcile this discrepancy, it was assumed that thermal activation and photon activation of rhodopsin follow different molecular mechanisms. The most convincing hypothesis for a separate low-energy thermal pathway is that the discrete dark noises of rods arise in a small subpopulation of rhodopsins, where the Schiff base linking the chromophore to the protein part has been deprotonated. According to Narici et al.’ experiments (2009, Radiation Measurements), phosphene perception in space travel is due to the ionizing radiation-induced free radicals that generate chemiluminescent photons from lipid peroxidation. These photons are absorbed by the photoreceptors chromophores, which modify the rhodopsin molecules (bleaching) and start the photo-transduction cascade resulting in the perception of phosphenes. Here, we point out that not only retinal phosphenes but also the discrete dark noise of rods can be due to the natural redox related (free radical) bioluminescent photons in the retina. In other words, under regulated conditions, lipid peroxidation is a natural process in cells and also in retinal membranes. Since the natural lipid peroxidation is one of the main sources of bioluminescent photons and the photoreceptors have the highest oxygen demand and polyunsaturated fatty acid (PUFA) concentration, there is a continuous, low level bioluminescent photon emission in the retina without any external photonic stimulation. During photopic or scotopic vision, evanescent bioluminescent photon emission is negligible. In contrast, in dark-adapted retinal cells this evanescent bioluminescent photon emission is not negligible. Therefore, our hypothesis is that the discrete dark noise of rods can be due to these bioluminescent photons.

γ-Radiation Induced Interstrand Cross-Links in PNA:DNA Heteroduplexes

Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that gamma-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e(-)(aq)) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, (*)OH, and reducing, e(-)(aq), radicals presents a case in which multiple ionization events along a gamma-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.

Radiomodifying and anticlastogenic effect of Zingerone on Swiss albino mice exposed to whole body gamma radiation

The radioprotective effect and antigenotoxic potential of phenolic alkanone, Zingerone (ZO) were investigated in Swiss albino mice exposed to gamma radiation. To study the optimum dose for radiation protection, mice were administered with ZO (10–100 mg/kg b.wt.), once daily for five consecutive days. One hour after the last administration of ZO on the fifth day, animals were whole body exposed to 10 Gy gamma radiations. The radioprotective potential was assessed using animal survival at an optimal ZO dose of 20 mg/kg b.wt., administered prior to 7–11 Gy. Further, the radioprotective potential of ZO was also analyzed by haemopoietic stem cell survival (CFU) assay, mouse bone marrow micronucleus test and histological observations of intestinal and bone marrow damage. Effect of ZO pretreatment on radiation-induced changes in glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPx) levels was also analyzed. ZO treatment resulted increase in the LD 50/30 by 1.8 Gy (dose reduction factor = 1.2). The number of spleen colonies after whole body irradiation of mice (4.5 or 7.5 Gy) was increased when ZO was administered 1 h prior to irradiation. The histological observations indicated a decline in the villus height and crypt number with an increase in goblet and dead cell population in the irradiated group, which was normalized by pretreatment with ZO. A significant ( p < 0.001) reduction in micronucleated polychromatic, normochromatic erythrocytes, increased PCE/NCE ratio, increase in the GSH, GST, SOD, CAT and decreased LPx levels were observed in ZO pretreated group when compared to the irradiated animals. Our findings demonstrate the potential of ZO in mitigating radiation-induced mortality and cytogenetic damage, which may be attributed to inhibition radiation-induced decline in the endogenous antioxidant levels and scavenging of radiation-induced free radicals.

Electron Paramagnetic Resonance Study of Radiation Damage in Photosynthetic Reaction Center Crystals

Electron paramagnetic resonance (EPR) was used to simultaneously study radiation-induced cofactor reduction and damaging radical formation in single crystals of the bacterial reaction center (RC). Crystals of Fe-removed/Zn-replaced RC protein from Rhodobacter ( R.) sphaeroides R26 were irradiated with varied radiation doses at cryogenic temperature and analyzed for radiation-induced free radical formation and alteration of light-induced photosynthetic electron transfer activity using high-field (HF) D-band (130 GHz) and X-band (9.5 GHz) EPR spectroscopies. These analyses show that the formation of radiation-induced free radicals saturated at doses 1 order of magnitude smaller than the amount of radiation at which protein crystals lose their diffraction quality, while light-induced RC activity was found to be lost at radiation doses at least 1 order of magnitude lower than the dose at which radiation-induced radicals exhibited saturation. HF D-band EPR spectra provide direct evidence for radiation-induced reduction of the quinones and possibly other cofactors. These results demonstrate that substantial radiation damage is likely to have occurred during X-ray diffraction data collection used for photosynthetic RC structure determination. Thus, both radiation-induced loss of photochemical activity in RC crystals and reduction of the quinones are important factors that must be considered when correlating spectroscopic and crystallographic measurements of quinone site structures.

EPR and UV investigation of sucrose irradiated with nitrogen ions and gamma-rays

Sucrose irradiated with N-ions and gamma-rays is investigated by EPR and UV spectroscopy. Irradiation doses are in the range of 20–380 Gy and linear energy transfer (LET) for N-ions of 90, 125 and 160 keV μ m - 1 . All EPR spectra recorded in irradiated solid samples are identical, suggesting that generated free radicals are not sensitive to the radiation quality. The EPR response of free radicals linearly depends on the absorbed dose but when generated by N-ions it is always lower than that obtained by irradiation with the same absorbed dose from gamma-rays. Water solutions of irradiated sucrose exhibit UV absorption at 267 nm due to the product of free radicals recombination, which spectrum remains independent on the radiation quality. The intensity of this band however depends on the absorbed dose and for equal doses it is higher for samples irradiated with N-ions than with gamma-rays. For different high LET-radiations with N-ions and low-LET radiation from gamma rays is found an excellent linearity between the EPR signal intensity and the UV absorbance of the obtained water solution. The relation between EPR and UV response of N-ions irradiated samples is reciprocal as well as between them and gamma-irradiated samples suggesting different impact of the high-energy photons and heavy particles on the solid state. For low LET photon radiation with moderate dose rate the probability for a successful hitting of two neighboring molecules is low. Therefore, only a small part of low LET radiation-induced free radicals may be expected to recombine giving an UV detectable product. In opposite, because of the dense ionizations and heaviness of N-ions almost all molecules in the core of the track are ionized yielding mainly products of the recombined free radicals. In the outer part of the track delta particles, which are more like low LET radiation electrons, stands for ionizations and produces a small number of free radicals situated far from each other. Therefore, we can expect higher EPR response of sugar irradiated with a certain dose of gamma-rays than of heavy particles. Just the opposite behavior should be expected for the UV absorption, which should be higher in the irradiation with heavy particles than with gamma-rays. The reported observations suggest that a sucrose/UV/EPR dosimetric system seems to be very promising for dose determinations from both gamma and heavy particle radiation and indicate a method for simultaneous determination of absorbed dose and LET. It also seems to be a new way for calibration of EPR dosimeters irradiated with N-ions via UV spectrometry.

Mechanisms of radiation-induced brain toxicity and implications for future clinical trials

Radiation therapy is widely used in the treatment of primary malignant brain tumors and metastatic tumors of the brain with either curative or palliative intent. The limitation of cancer radiation therapy does not derive from the inability to ablate tumor, but rather to do so without excessively damaging the patient. Among the varieties of radiation-induced brain toxicities, it is the late delayed effects that lead to severe and irreversible neurological consequences. Following radiation exposure, late delayed effects within the CNS have been attributable to both parenchymal and vascular damage involving oligodendrocytes, neural progenitors, and endothelial cells. These reflect a dynamic process involving radiation-induced death of target cells and subsequent secondary reactive neuroinflammatory processes that are believed to lead to selective cell loss, tissue damage, and functional deficits. The progressive, late delayed damage to the brain after high-dose radiation is thought to be caused by radiation-induced long-lived free radicals, reactive oxygen species, and pro-inflammatory cytokines. Experimental studies suggest that radiation-induced brain injury can be successfully mitigated and treated with several well established drugs in wide clinical use which exert their effects by blocking pro-inflammatory cytokines and reactive oxygen species. This review highlights preclinical and early clinical data that are translatable for future clinical trials.

Differential diagnosis of Acute Radiation Syndromes by Enzyme Immune-Assay (EIA)

Differential diagnosis of Acute Radiation Syndromes by the method of immune enzyme assay is a very efficient tool of biological dosimetry and evaluation of acute radiation disease. We use as biological markers the group of essential Radiotoxins - high molecular weight glycoproteins with specific antigenic properties. A molecular weight of radiation toxins was 200-250 kDa. High doses of radiation induce the formation of radiation toxins in the organs and tissues of irradiated animals. After whole body irradiation, cellular macromolecules and cells and mitochondrial outer and inner wall membranes are damaged by long-lived radiation-induced free radicals, reactive oxygen species and fast, charged particles of radiation. High doses of radiation induce breaks in the chemicals bonds of macromolecules (proteins,lipids,carbohydrates,DNA molecules) and cross-linking reactions via chemically active processes. These processes result in creation of novel modified macromolecules that possess specific toxic and antigenic properties defined by the type and doses of irradiation by which they are generated. After formation, Radiation toxins migrate from irradiated tissues to interstitial fluid and accumulate in the lymphatic and blood transportation system. Radiation toxins isolated from the lymph of irradiated animals are classified as hematotoxic, neurotoxic, and enteric non-bacterial(GI) Radiation Toxins, and they play an important role in development of Hematopoietic, Cerebrovascular, Cardiovascular and Gastro- Intestinal Acute Radiation Syndromes(ARS). Four Groups and Seven distinct Toxins derived from post-irradiated animals have been designated as Specific Radiation Determinants (SRD): SRD-1(Neuro-toxic radiation toxin generated by Cerebrovascular ARS), SRD-2(Vascular-toxic radiation toxin generated by Cardiovascular ARS), SRD-3(Enteric non-bacterial radiation toxins generated by the Gastrointestinal form of ARS), and SRD-4(Hematotoxic radiation toxins generated by hematological, bone marrow form of ARS ). SRD-4 is further subdivided into four groups depending on the severity of the ARS induced: SRD-4/1, mild ARS; SRD-4/2, moderate ARS; SRD-4/3, severe ARS and SRD-4/4, extremely severe ARS. We postulate that the SRD-1 and the SRD-2 radiation toxins produce toxicity for central and peripheral nervous system. Determination of high levels of SRD-1, SRD-2,SRD-3 and SRD-4 in the periferal blood allowed to recognize early periods of Cerebrovascular, Cardiovascular, Gastrointestinal and Hematopoietic forms of ARS. The important goal of an early assessment with Enzyme Immune Assay is the accurate description of the Acute Radiation Syndromes at initial phases. Early and precise differential diagnosis allow doctors to provide an effective medical management of ARS.

Irradiated lithium formate and amino acids

Radiation-induced free radicals in solids show a microwave saturation effect when studied by electron spin resonance. A comparison is made between such effects in lithium formate and amino acids. The relative effectiveness of neutrons against high-energy photons is also considered.

The effect of pre-irradiation vacuum storage on the oxidation and wear of radiation sterilized UHMWPE

The adverse biological response to ultra-high molecular weight polyethylene (UHMWPE) wear debris in total joint replacement warrants the need for the material's improved wear resistance. The reaction of oxygen with radiation-induced free radicals within UHMWPE leads to oxidative degradation of the material. This work examines the effect of storing UHMWPE in vacuum at an elevated temperature (70 °C) prior to gamma irradiation in an attempt to remove pre-existing free oxygen from within the material and hence reduce oxidation that occurs during the irradiation process. UHMWPE specimens both with and without pre-irradiation vacuum were gamma irradiated to either 4 or 10 Mrad and then artificially aged to simulate the material's long-term oxidative behavior. Oxidation, degree of crosslinking, and wear resistance were measured. The benefit of pre-irradiation vacuum in terms of reduced oxidation, higher degree of crosslinking, and improved wear resistance measured after aging was observed for both 4 and 10 Mrad irradiation doses.

Reactive species and apoptosis of neural precursor cells after γ-irradiation

This study addresses the participation of radiation-induced free radicals, mainly nitric oxide (NO), in modulating the apoptotic response in an in vitro model of neural cortical precursor cells exposed to γ-radiation. Cortical cells obtained from rats at 17 gestational day (GD) were irradiated with a dose of 2 Gy. The percentage of apoptotic cells was significantly increased 4 h post-irradiation (pi). NO content showed a significant increase after 30 min pi and the rate of generation reached a maximum 1 h pi. Luminol-dependent chemiluminescence (CL) was significantly higher in cells after 2 h pi as compared to control cells and this profile was maintained up to 4 h pi. Supplementation with l-NAME significantly increased light emission. Administration of superoxide dismutase (SOD) following l-NAME addition prevented the observed changes due to l-NAME administration. The caspase inhibitor zDEVD-fmk significantly reduced the radical generation. Moreover, the cellular decrease in NO content occurred coincidentally with the rise in oxygen radical generation and the activation of caspase-3. In vitro irradiation of neural precursor cells allowed us to suggest that an early radiation-induced generation of NO could exert a neuroprotective role. However, despite this NO initial protective effect and its role modulating the response against γ-radiation, NO generation was not able of fully preventing radiation-induced apoptosis.

Thermal stability of radiation-induced free radicals in [formula omitted]-irradiated l-alanine single crystals

Decay of the radiation-induced stable free radicals in l-alanine single crystals and powders at the temperatures from 379 to 476 K was examined by electron paramagnetic resonance. For single crystals, the calculated activation energy of the radical decay is 104.3±1.7 kJ/mol (i.e. 12 538±202 K) and the frequency factor ln ν 0 is 24.1±0.4 min −1. The lifetime of the radical in single crystals at 296 K is 162 years. The results confirm the long-term stability of the radicals, but the decay was found to be faster in large crystals than in powders.

The Solid‐State Radiation Chemistry of Simple Amino Acids ( Revisited)

AbstractFor Abstract see ChemInform Abstract in Full Text.

The solid-state radiation chemistry of simple amino acids, revisited.

The solid-state radiation-induced free radical formation in simple amino acids like alpha-glycine (gly) and L-alpha-alanine (ala) has been the subject of investigations by EPR spectroscopy since the late 1950s. The EPR spectra from crystals of gly and ala generally are very complex due to the simultaneous trapping of several free radicals regardless of irradiation and observation temperatures. Untangling these complex spectra is necessary for understanding the mechanisms for the solid-state radiation chemistry of amino acids. Recently, radical formation in gly and ala after room-temperature irradiation has been reinvestigated in our laboratories using X-, K- and Q-band EPR and ENDOR spectroscopy, combined with the ENDOR-induced EPR (EIE) techniques as well as single-crystal and powder EPR and ENDOR spectrum simulations. Several new radical products have been detected and characterized, most prominently the gly species H2N - C x H - COOH and the ala species H3+N - C x (CH3) - COO and H2N - C x (CH3) - COOH. A short description of these radicals is given, and an overview of the solid-state radiation chemistry of the simple amino acids is presented, based on a review of the literature combined with these recent experimental results.

Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry

By use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, phosphatidic acid was found to be the main product of γ radiolysis of cardiolipin, phosphatidylinositol, and phosphatidylglycerol. It has been shown that γ irradiation of such glycolipids as cerebroside and galactosyl diglyceride leads to formation of ceramide and diglyceride, respectively. These findings, combined with those obtained earlier, allowed an assumption to be made that, owing to radiation-induced free radical fragmentation of lipids in their polar moiety, formation of signaling molecules can occur.

Experimental and Theoretical Electron Magnetic Resonance Study on Radiation-Induced Radicals in α-l-Sorbose Single Crystals

α-l-Sorbose single crystals were X-irradiated at 295 K (room temperature). A combined electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EI-EPR) study at 120 K revealed a realm of radiation-induced free radicals in this sugar system. In the present work, a pair of closely related radicals is focused on, being dominant immediately after irradiation, but unstable with respect to long time storage or upon warming the samples. A density functional theory (DFT) study was carried out considering the complete hyperfine coupling tensors (principal axes and anisotropic and isotropic couplings) in comparison with the observed electron−proton interactions. This combined approach yielded very plausible models for both radicals, which are formed by a net hydrogen-abstraction from the C3 position of the six-membered sorbose ring. It appears that the difference between the two species is linked to the molecular disorder in the sorbose crystal structure. In addition, DFT calculations of the g tensors were performed for the plausible radical conformations.

The ESR spectroscopic features and kinetics of the radiation-induced free radicals in maize ( Zea mays L.)

A detailed ESR investigation on the characteristic features and kinetic behaviors at different temperatures of the long-life free radicals induced in gamma irradiated ground maize cultivated in Turkey was carried out in the dose range of 0–5.0 kGy. Unirradiated ground maize exhibits a weak, broad single-line ESR spectra centered at g=2.0048 originating from a radical species of unknown structure called as radical III in this work. Irradiation produced two more radicals called radical I and radical II, beside radical III. The radicals (I, II and III) follow complicated kinetics. Room and high temperatures signal intensity decay data along with data derived from room temperature ESR spectra were used together to calculate spectroscopic features of the contributing radicals. A model based on three radicals of different features found to describe best experimental results obtained for irradiated maize. Dosimetric properties of maize in the studied dose range were also reported.

Electron Spin Resonance Studies on Electron Beam Irradiated Carbon Nanotubes Dispersed in Styrene-Isoprene-Styrene Block Copolymer

ABSTRACTElectron spin resonance investigations on the effect of electron bombardment of nanocomposites obtained by dispersing carbon nanotubes within styrene-isoprene-styrene are reported. The experimental results revealed the absence of radiation-induced free radicals and a negligible effect of electron beam irradiation on the electron spin resonance spectra of carbon nanotubes.

Determination of the activation energies of and aggregate rates for exothermic physico-chemical changes in UHMWPE by isothermal heat-conduction microcalorimetry (IHCMC)

Exothermic heat flow rates ( Q=μW=μJ/s), as a function of elapsed time, were measured by isothermal heat-conduction microcalorimetry (IHCMC) in order to study the aggregate rate of physico-chemical change in specimens of unsterilized and sterilized ultra-high-molecular-weight polyethylene (UHMWPE). Standard protocols for performing the IHCMC tests were developed and are described. Use of the standard protocols yielded the desired results—data that were not significantly different among either replicate sets of unsterilized specimens or as a function of which calorimeter test well was used. Heat flow rates measured in air at 20°C, 25°C, 35°C, and 45°C yielded estimates of activation energies of 47, 11, and 41 kJ/mol for unsterilized, γ-radiation sterilized, and ethylene oxide gas (EtO) sterilized polymer, respectively. These results support the ideas that (a) initial exothermic degradation takes place much more easily in the radiation-sterilized material, due to direct oxidation of readily available free radicals, and (b) the much slower degradation process in EtO-sterilized UHMWPE is not appreciably different than in unsterilized polymer. Comparison with other activation energy data suggests that the rate-limiting process in EtO- or un-sterilized polymer is oxygen diffusion into the polymer. For shelf storage in air, for periods up to 8 months, the mean exothermic heat flow in air, at 25°C ( Q m) [determined from the Q values averaged over the time period between 15 and 20 h after test start], from UHMWPE γ-radiation sterilized in air was significantly higher than for unsterilized material (2.91±0.11 vs. 0.73±0.11 μW). The higher rate can be attributed to oxidation of radiation-induced free radicals in the polymer near its surface. For the γ-irradiated polymer, the decline in Q m with shelf storage time suggests that, eventually, degradation might become oxygen diffusion limited in this case also. However, in vivo, surface wear of an UHMWPE articular component may continue to expose unoxidized free radicals, keeping the exothermic reaction rate high and, possibly, continuing to produce an oxidized UHMWPE surface prone to wear.