The effect of dose on light-sensitivity of radicals in alanine EPR dosimeters

In this work we present a study of light-induced effects on free radicals and their transformations in gamma-irradiated pure L-alanine and in commercially available alanine detectors: rods, pellets and films. Samples irradiated to doses from 2 Gy to 4000 kGy were exposed to light from a fluorescent lamp and to ordinary daylight. The observed changes in EPR spectra of the samples were analyzed with regard to their intensity and shape. The shape analysis was based on numerical decomposition of the measured spectra into model spectra reflecting contributions of R1, R2 and R3 radical populations in the samples. The illumination of alanine dosimeters resulted in significant decrease of the central EPR line and was accompanied by distinct variations in the shape of EPR spectra. The rate of light-induced decay in spectra amplitude was found to be dependent on dose of ionizing radiation--the sensitivity to light was decreasing with increase in dose in all detectors in the 2-5x10(5) Gy dose range. The exposure of gamma-irradiated (to 300 Gy) alanine to normal, diffused daylight resulted in decay of the signal amplitude at rate about 0.5% per week. It was shown that decay in the R1 component was responsible for the observed reduction of the spectra amplitude. The observed increase in R2 contributions in samples exposed to light confirmed a hypothesis of R-->R2 radical transformations promoted by visible light. The reported effects indicate a necessity of protection of irradiated dosimeters from their prolonged exposure to light.     

Decomposition study of the electron paramagnetic resonance spectrum of irradiated alanine

Recent Electron Paramagnetic Resonance (EPR) studies on alanine powders as a function of irradiation dose and temperature on the one hand and single crystal Electron Nuclear DOuble Resonance (ENDOR) studies on the other hand, showed the presence of at least three radicals contributing to the total alanine EPR spectrum. The latter spectrum obtained after irradiation at room temperature (RT), is dominated by the well-known stable-alanine-radical (SAR) CH3C*HCOO-, also denoted R1. Appropriate heating of irradiated alanine causes the relative contribution of R1 to decrease, resulting in a spectrum mainly caused by the H-abstraction radical CH3C*(NH3)COO-, denoted R2. Although the EPR spectrum of these two radicals could be satisfactorily simulated, their influence on dose reconstruction has not been reported yet. Therefore, a detailed Maximum Likelihood Common Factor Analysis (MLCFA) study has been performed on EPR spectra from polycrystalline alanine samples, after irradiation and heat treatments. Conclusions concerning the number of contributing radicals and their influence on the RT irradiated alanine EPR spectrum will be made.     

EPR PERSISTENCE MEASUREMENTS OF UV-INDUCED MELANIN FREE RADICALS IN WHOLE SKIN

Electron paramagnetic resonance is used to detect the formation of free radicals caused by exposure to ultraviolet radiation in chemically untreated rabbit skin. A fast jump in EPR signal level, occurring over a few seconds, is observed immediately after a skin sample is exposed to UV. This is followed by a slower increase toward an elevated steady-state signal over a period of hours as the skin is continuously exposed to a UV light source. Upon cessation of UV light exposure, EPR signal levels undergo an abrupt drop followed by a slower decay toward natural levels. Elevated free radical concentrations following UV exposure are found to persist for several hours in whole skin. These results are consistent with time-resolved EPR measurements of photoinduced radicals in various natural melanins.     

ULTRAVIOLET LIGHT-INDUCED FREE RADICAL FORMATION IN SKIN: AN ELECTRON PARAMAGNETIC RESONANCE STUDY

It has been suggested that ultraviolet light induces free radical formation in skin, leading to photoaging and cancer. We have demonstrated by electron paramagnetic resonance that the ascorbate free radical is naturally present in unexposed skin at a very low steady state level. When a section of SKH-1 hairless mouse skin in an EPR cavity is exposed to UV light (4,500 J m⁻²−1, Xe lamp, 305 nm cutoff and IR filters), the ascorbate free radical signal intensity increases. These results indicate that UV light increases free radical oxidative stress, consistent with ascorbate's role as the terminal, small-molecule antioxidant. The initial radicals produced by UV light would have very short lifetimes at room temperature; thus, we have applied EPR spin trapping techniques to detect these radicals. Using α-[4-pyridyl 1-oxide]-N- tert -butyl nitrone (POBN), we have for the first time spin trapped a UV light-produced carbon-centered free radical from intact skin. The EPR spectra exhibited hyperfine splittings that are characteristic of POBN/alkyl radicals, a^N= 15.56 G and a^H= 2.70 G, possibly generated from membrane lipids as a result of β-scission of lipid alkoxyl radicals. Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron-chelator, Desferal, to a section of skin reduces the UV light-induced POBN adduct radical signal. These results provide direct evidence for free radical generation and a role for iron in UV light-induced dermatopathology. We suggest that iron chelators can serve as photoprotective agents by preventing these oxidations.     

ELECTRON AND ENERGY TRANSFER IN ILLUMINATED POROUS VYCOR GLASS

Abstract— Porous Vycor glass samples containing adsorbed molecules were illuminated at 77 K by a mercury lamp jacketed by a filter cutting off wavelengths below 250 nm. Oxygen or carbon dioxide on Vycor produces an asymmetric electron paramagnetic resonance (EPR) signal best described as holes trapped in the glass. Methyl bromide produces an identical EPR signal plus four other lines due to methyl radicals. Evidence is presented that the products result from excitonic energy transfer from the Vycor to the adsorbed materials. Triphenylamine (TPA) adsorbed on Vycor can also be photoionized by similar illumination, and the cation radical TPA⁺ can be stabilized at 77 K if an electron acceptor is also adsorbed. Attachment of the photoejected electron by carbon dioxide forms CO₂^-, and that by methyl bromide leads to methyl radicals. The CH₃ radical yield is dependent on the surface separation between the electron donor (TPA) and the acceptor (CH₃Br). By monitoring the relative quantum yield of the methyl radicals as a function of distance separating the TPA and CH₃Br, it is shown that the photoelectron is capable of migrating on the Vycor glass surface.     

Radical kinetics and characterization of the free radicals in gamma irradiated red pepper

Kinetic behaviors and characterization of the natural and γ-induced radicals in irradiated red pepper have been investigated by electron spin resonance (ESR) spectroscopy to explore the possibility of using this technique in detecting irradiated red pepper and to evaluate the eventual dosimetric features of this widely used food. Unirradiated samples exhibited a single resonance line centered at g=2.0050±0.0005. Photo-exposure of the samples was found to increase the signal intensity. An increase in temperature created a drastic decrease in the concentration of natural radicals responsible for the single resonance line. Irradiation was observed to induce increases in the intensity of single resonance line (signal I) and a radiation specific doublet and/or triplet (signal II) also centered at g=2.0050 but detectable only at high spectrometer gains. The intensities of both signals increased with increasing radiation dose. The signals I and II were found to decay with different rates depending on the temperature. The results of a fitting procedure applied to the experimental signal decay curves and those obtained from room temperature spectra simulation calculations were used together to determine radical species and their spectral characteristics giving rise to the observed experimental spectra. Four radical species, three carbohydrate and one semiquinone radical assigned as radicals A, B, C and D, respectively, were found to best explain the experimental results. All the radicals show large g and hyperfine splitting anisotropies varying between g=2.0028–2.0062 and 1.07–2.58 mT, respectively. The half lives of the radicals were found to depend strongly on temperature. The activation energy calculated using temperature dependent half-life data were the highest for radical A (33.68 kcal/mol) and smallest for radical C (11.83 kcal/mol).     

Formation of free radicals in photoirradiated cellulose. V. Effect of temperature

ESR spectra of purified and ferric ion-sensitized celluloses irradiated with ultraviolet light in vacuo at 45, 20, ?80, and ?196°C were recoreded and compared. Generally, several kinds of spectra, viz., singlet, three-line, five-line, and seven-line spectra, were observed. At higher temperatures, only singlet and three-line spectra of stable free-radical species were detected, whereas at lower temperatures such as at ?196°C, two doubled spectra of formyl radicals and hydrogen atoms were also detected in addition to cellulose radicals. It is believed that the intricate spectra observed at low temperatures are superimposed upon spectra generated by free radicals which may or may not be stable at high temperatures. During reirradiation at ?196°C with an alternative light sources, i.e., λ > 2537 Å and λ > 3400 Å, of samples which were irradiated at 20°C or at ?196°C, phenomena indicative of radical transformation and formation of new radicals or of decay of radicals in terms of ultraviolet bleaching were observed on studying the changes of line-shapes and relative signal intensities of the spectra.     

Fourier transform-EPR studies of photoinduced electron transfer in homogeneous and micellar solutions

Fourier transform (FT) EPR was used to study the pulsed-laser-induced electron transfer from porphyrins to quinones in homogeneous and micellar solutions. By monitoring the EPR signal of the quinone anion radicals as a function of delay time (τ_d) between laser and microwave pulses, with τ_d ranging from nanoseconds to 1 millisecond, information was obtained on the kinetics of free radical formation and decay. The time evolution of the signal also gave an insight into the chemically induced dynamic electron polarization (CIDEP) mechanisms that affect signal amplitudes. FT-EPR spectra of electron transfer products generated in micellar solution provide evidence for the generation of long-lived spincorrelated radical pairs.     

The oxidation and thermal transformations of macrooradicals in gamma irradiated cellulose

The secondary reactions of the oxidation and thermal transformations of gamma irradiated (at 77 K) and plasticized (with water) cellulose radicals were studied by 3 cm-and 2 mm-band EPR spectroscopy. The radiolysis of cotton cellulose was found to produce the H-C*=O formyl radical, and heating the irradiated samples to 190–200 K resulted in the formation of the ROO* peroxide radical. The EPR spectra of microcrystalline cellulose recorded at room temperature contained an individual triplet (α _β ^H = 2.5–2.7 mT) with an additional quadruplet structure (splitting 0.5–0.7 mT) from three γ-hydrogens. This triplet was interpreted as a signal of the primary radical at C4. The main direction of thermal transformations of primary radicals was synchronous reactions of the dehydration of the polycarbohydrate complex accompanied by the dissociation of the C-H, C-OH, and C-C bonds and elimination of H₂O, H₂, CO, and CO₂ with successive formation of allyl and then polyene radicals, which were a source of the growth of polyconjugated systems in macromolecules.     

ON THE PHOTOCHEMISTRY OF X-RAY PRODUCED RADICALS TRAPPED IN FROZEN ETHANOL MATRICES

Abstract— Ethanol and ethanol-water matrices were exposed to X-rays at 77K and the photochemistry and paths of radical conversion were investigated by EPR methods. The main X-ray induced radical, CH3ĊHOH, is probably photoionized by 254 nm light. The following radicals are produced during prolonged UV-irradiation of CH₃ĊHOH radicals: ĊH₃, ĊHO, H and 2 types of radicals giving singlet EPR spectra. One of these radicals (d) is bleachable with 580 nm light, ĊH₃ and ĊH₃ĊHOH being formed during the bleaching, the other one (e) is unbleachable and the most stable radical in the matrix during annealing. The CH₃ radicals decay at 77 K (τ∽ 10 min) and produce CH₃-CHOH radicals and the unbleachable radical (e). Stable H-atom signals were seen in X-irradiated ethanol-water mixtures (volume ratio 2:1) at 77 K. The H-atom signals increased during photobleaching of the trapped electrons in the matrix and during UV-photolysis of CH₃CHOH radicals.     

Identification of primary free radicals in trehalose dihydrate single crystals X-irradiated at 10 K

Primary free radical formation in trehalose dihydrate single crystals X-irradiated at 10 K was investigated at the same temperature using X-band Electron Paramagnetic Resonance (EPR), Electron Nuclear Double Resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques. The ENDOR results allowed the unambiguous determination of six proton hyperfine coupling (HFC) tensors. Using the EIE technique, these HF interactions were assigned to three different radicals, labeled R1, R2 and R3. The anisotropy of the EPR and EIE spectra indicated that R1 and R2 are alkyl radicals (i.e. carbon-centered) and R3 is an alkoxy radical (i.e. oxygen-centered). The EPR data also revealed the presence of an additional alkoxy radical species, labeled R4. Molecular modeling using periodic Density Functional Theory (DFT) calculations for simulating experimental data suggests that R1 and R2 are the hydrogen-abstracted alkyl species centered at C5' and C5, respectively, while the alkoxy radicals R3 and R4 have the unpaired electron localized mainly at O2 and O4'. Interestingly, the DFT study on R4 demonstrates that the trapping of a transferred proton can significantly influence the conformation of a deprotonated cation. Comparison of these results with those obtained from sucrose single crystals X-irradiated at 10 K indicates that the carbon situated next to the ring oxygen and connected to the CH(2)OH hydroxymethyl group is a better radical trapping site than other positions.     

The effect of heating on background and radiation-induced EPR signals in tooth enamel

The presented study is a continuation of our work performed during participation in the Third International Intercomparison on EPR Tooth Dosimetry. During the process of samples preparation, all 22 enamel samples were accidentally exposed for about 30 min to 150 degrees C temperature. This considerably affected shape of their EPR spectra mainly due to substantial increase in the background signal, which approximately doubled its contribution to the spectra. These effects were studied closer under controlled conditions of the delivered dose and heating temperature using another enamel samples. The observed changes in the spectra shape partially faded within a few days after heating. The heating resulted also in a noticeable generation of a spectral component similar to the dosimetric signal induced in enamel by radiation. The temperature-induced dosimetric component in EPR spectra of the heated samples remained constant for 32 days. Deviations in calculated contributions of the dosimetric signal into total EPR spectra of irradiated sample varied from -12 to +15% of its initial contribution in the non-heated enamel, depending on type of the background spectrum applied in numerical processing of the spectra.     

A DNA oligonucleotide-hemin complex cleaves t-butyl hydroperoxide through a homolytic mechanism

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex ( approximately 35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of approximately 250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of < or =5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from beta-scission of the primary tert-butyloxyl radical product.     

Simplification of complex EPR spectra by cepstral analysis

As the Fourier transform of time-series data is known as the spectrum, the Fourier transform of the logarithm of the time-series data is called the cepstrum of the data. When cepstral analysis is applied to free induction decay signals of free radicals showing first-order EPR spectra, the identification of nuclear hyperfine coupling constants becomes simple. In a systematic manner, we have examined how the technique of cepstral analysis is affected by the presence of aliasing, noise, uncertainty in the time origin of the free induction decay, the presence of second-order hyperfine couplings, and the applications of various apodization methods. This technique was then applied to analyze the EPR spectrum of anthraquinone anion radical, and anion radicals of porphycene and tetrapropyl-porphycene, and the hyperfine coupling constants thus obtained were compared with published data. A good agreement was always found. We make a case for the usefulness of cepstral analysis in determining the hyperfine coupling constants of complex EPR spectra of organic free radicals.     

Characterization of an LED based photoreactor to degrade 4-chlorophenol in an aqueous medium using coumarin (C-343) sensitized TiO2

A detailed performance evaluation of a simple high intensity LED based photoreactor exploiting a narrow wavelength range of the LED to match the spectrum of a dye in a photocatalysis system is reported. A dye sensitized (coumarin-343, lambda max = 446 nm) TiO 2 photocatalyst was used for the degradation of 4-chlorophenol (4-CP) in an aqueous medium using the 436 nm LED based photoreactor. The LED reactor performed competitively with a conventional multilamp reactor and sunlight in the degradation of 4-CP. Light intensities entering the reaction vessel were measured by conventional ferrioxalate actinometry. The results can be fitted by approximate first order kinetic behavior in this system. Hydroxyl radicals were detected by spin trapping EPR, and effects of OH radical quenchers on kinetics suggest that the reaction is initiated by these radicals or their equivalents. LEDs operating at competitive intensities offer a number of advantages to the photochemist or the environmental engineer via long life, efficient current to light conversion, narrow bandwidth, forward directed output, and direct current power for remote operation. Matching light source spectrum to chromophore is a key.     

Relevance of sunscreen application method, visible light and sunlight intensity to free-radical protection: A study of ex vivo human skin

With the continued rise in skin cancers worldwide there is a need for effective skin protection against sunlight damage. It was shown previously that sunscreens, which claimed UVA protection (SPF 20+), provided limited protection against UV-induced ascorbate radicals in human skin. Here the results of an electron spin resonance (ESR) investigation to irradiate ex vivo human skin with solar-simulated light are reported. The ascorbate radical signal in the majority of skin samples was directly proportional to the irradiance over relevant sunlight intensities (0.9-2.9 mW cm(-2)). Radical production (substratum-corneum) by UV (wavelengths < 400 nm) and visible components (> 400 nm) was approximately 67% and 33% respectively. Ascorbate radicals were in steady state concentration at low irradiance (approximately 1 mW cm(-2) equivalent to UK sunlight), but at higher irradiance (approximately 3 mW cm(-2)) decreased with time, suggesting ascorbate depletion. Radical protection by a four star-rated sunscreen (with UVA protection) was optimal when applied as a thin film (40-60% at 2 mg cm(-2)) but less so when rubbed into the skin (37% at 4 mg cm(-2) and no significant protection at 2 mg cm(-2)), possibly due to cream filling crevices, which reduced film thickness. This study validates ESR determinations of the ascorbate radical for quantitative protection measurements. Visible light contribution to radical production, and loss of protection when sunscreen is rubbed into skin, has implications for sunscreen design and use for the prevention of free-radical damage.     

Study of the gamma irradiation effects on the PMMA/HA and PMMA/SW

The behavior of the poly(methyl methacrylate) (PMMA) under the action of gamma radiation has been sufficiently studied. In this work, we present results from melt flow index (MFI), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) of PMMA composites with hydroxyapatite (HA) and seaweed residues (SW) irradiated with gamma rays at 1.08 kGy/h. Composites of PMMA/HA and PMMA/SW with 10%, 20% and 30% of the filler were prepared. The results show an increase in the MFI values with the integral dose of radiation, being consistent with chain-scission reactions. No EPR signal was observed in pure PMMA, while in the composites, the typical EPR signal of the PMMA radicals was observed, which increased with the amount of HA or SW. When comparing the relative intensities of the EPR signals for both types of composites, a slight increase in the concentration of free radicals generated in the sample with SW respect to that of PMMA/HA composite was obtained. A decay of the total free radical concentration was observed as time elapsed.     

Comparative decay characteristics of the light generated free radical in chromatophores and chloroplasts

Abstract— Chromatophores of Rhodospirillum rubrum when illuminated exhibit a free radical which ordinarily shows a biphasic decay. When chromatophores are dehydrated at room temperature the time course of the appearance of the light-induced free radical is unaffected, but the decay pattern has been altered. Only the fast component remains, the slow component is no longer evident. Scanning the magnetic field reveals the presence of a dark signal which is stable as long as the preparation remains dehydrated. This signal has the same peak-to-peak line width of ? 10 G and the same g factor as the signal evident in the light. The amplitude of this signal is equal to the amplitude of the slow decay component seen in aqueous chromatophore suspensions. Chromatophores frozen in an aqueous medium at —150°C exhibit a behavior identical with dehydrated preparations. The effects produced by lyophilization or by freezing at low temperatures are entirely reversible. When a lyophilized preparation is re-hydrated, the stabilized portion of the signal now decays in the dark; the same is observed when preparations frozen at —150°C are thawed. When such thawed or re-hydrated preparations are illuminated again, they exhibit the usual light-induced ESR signal showing a biphasic dark decay. A comparison was made between the behavior of the light-induced ESR signal of chromatophores and that of system I of chloroplasts. This comparison revealed that there is a greater similarity in some of the decay characteristics of these signals than had been recognized previously. In chloroplasts, temperature insensitive, non-enzymatic back-reactions of the light-induced free radical appear to be nil, and in chromatophores a distinct portion of the light-induced free radicals exhibit the same characteristics. Another portion of the chromatophore free radicals must be able to back-react by electron tunneling, a mechanism which appears to be absent in the chloroplast system.     

EPR study of melanin-protein interaction: photoinduced free radicals and progressive microwave power saturation.

The photo-induction of free radicals in synthetic L-dihydroxyphenylalanine (L-DOPA) melanin in the presence of bovine serum albumin (BSA) was studied by electron paramagnetic resonance (EPR) spectroscopy. By monitoring the signal intensities and progressive microwave power saturation it was shown that L-DOPA melanin in solution behaves as a single macromolecule, interacting with BSA and molecular oxygen. In the absence of oxygen, the EPR signal of L-DOPA melanin was homogeneously broadened; the magnetic interaction with oxygen induced inhomogeneous broadening. In aqueous solution, the presence of BSA decreased the accessibility of oxygen to paramagnetic centres in the melanin. On UV-visible illumination, the presence of BSA modified the rates of formation and decay of photoinduced free radicals, resulting in a net enhancement of the EPR signal compared with that observed in pure L-DOPA melanin.     

LOW COST DIGITAL SPECTRORADIOMETER*

Abstract— A self-made low cost digitized single beam spectroradiometer is described. It is designed and calibrated to measure spectral radiant intensities in one nanometer steps for bandwidths between 1 and 20 nm within the wavelength range 200–1100 nm. The voltage output of the photoelectric detector-system is proportional to the logarithm of the received irradiance and so constant signal resolution is gained over more than three orders of radiant intensities. Raw data recording and processing is done by a common 8 bit microcomputer, programmed in Microsoft Basic. Scanned emission spectra of a far-red fluorescent lamp and a long arc xenon discharge lamp are presented and compared to manufacturer's data. In addition, the emission spectrum of a blue fluorescent tube has been determined to test for photometric aptitude. In this context the integral irradiances have been both measured and calculated for three radiometric sensors of defined spectral responsivities, indicating reliability of the instrumentation.