Steven Swarts

An ESR Investigation of the Reactions of Glutathione, Cysteine and Penicillamine Thiyl Radicals: Competitive Formation of RSO·, R·, RSSR, and RSS·

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.

Antioxidants Reduce Consequences of Radiation Exposure

Antioxidants have been studied for their capacity to reduce the cytotoxic effects of radiation in normal tissues for at least 50 years. Early research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio, even though these compounds have substantial toxicity when given in-vivo. Other antioxidant molecules (small molecules and enzymatic) have been studied for their capacity to prevent radiation toxicity both with regard to reduction of radiation-related cytotoxicity and for reduction of indirect radiation effects including long-term oxidative damage. Finally, categories of radiation protectors that are not primarily antioxidants, including those that act through acceleration of cell proliferation (e.g. growth factors), prevention of apoptosis, other cellular signaling effects (e.g. cytokine signal modifiers), or augmentation of DNA repair, all have direct or indirect effects on cellular redox state and levels of endogenous antioxidants. In this review we discuss what is known about the radioprotective properties of antioxidants, and what those properties tell us about the DNA and other cellular targets of radiation.

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays--a measure of ATP production--under rapid phosphorylation conditions to explore sites of Mn(2+) inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn(2+) inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn(2+) inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F₁F₀ ATP synthase. In mitochondria fueled by either succinate or glutamate+malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn(2+) inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Mitigation Effect of an FGF-2 Peptide on Acute Gastrointestinal Syndrome after High-Dose Ionizing Radiation

PURPOSE Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS The study data support pursuing FGF-P as a mitigator for AGS.

An esr study of radical intermediates formed by γ - radiolysis of tripalmitin and dipalmitoyl phosphatidylethanolamine

Radicals resulting from the 7-radiolysis at 77 K of neat tripalmitin and neat dipalmitoyl phosphatidylethanolamine were investigated by electron spin resonance spectroscopy (ESR). Analyses of the spectra of these complex lipids were aided by ESR studies of their components phosphorylethanolamine, palmitic acid and hexa-decane. Concentrations of various radicals were followed as a func-tion of temperature after γ-irradiation at 77 K. Both tripalmitin and dipalmitoyl phosphatidylethanolamine show anion radicals formed by electron addition to the ester groups and aliphatic side-chain radicals (-CH2-CH-CH2-) which are thought to be the result of deprotonation of the original positive hole. Computer analyses of the spectra as the temperature increases show loss of the anion radicals with an increase in a new radical produced by the abstrac-tion of a hydrogen atom from the α carbon on an acyl side chain. In the case of dipalmitoyl phosphatidylethanolamine, a radical of the phosphorylethanolamine portion of the molecule is detected as an intermediate. Mechanisms for the formation and decay of the free radical intermediates are discussed.

B1 Sequence-based real-time Quantitative PCR: A sensitive method for direct measurement of mouse plasma DNA levels after gamma irradiation

PURPOSE Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. METHODS AND MATERIALS Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ((137)Cs gamma-ray source at approximately 1.86 Gy/min; homogeneity: +/- 6.5%). RESULTS The correlation coefficient between DNA levels and the threshold cycle value (C(T)) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples. CONCLUSIONS A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

An electron spin resonance investigation of ester cation radicals at low temperatures. Proton transfer and rearrangement reactions

The cation radicals of a series of esters have been produced by γ-irradiation of CFCl3 matrices containing the ester at 77 K. In previous work cations of methyl and ethyl esters were investigated. In this work we report results for larger esters. The cations of these esters are found to undergo immediate internal proton-transfer reactions involving specific sites on one of the alkyl substituents. For example, deuteration studies show that proton transfer occurs from the terminal methyl group in propyl formate to produce —OCH2CH2CH·2, whereas in propyl acetate —OCH2ĊHCH3 is produced. The proton lost from these groups is assumed to add to an oxygen on the ester functional group. In the case of propyl acetate and esters with branched side chains we find that fragmentation reactions follow the proton transfer. In the cases of t-butyl acetate and isobutyl formate the fragmentation process occurs at 77 K and results in the isobutylene cation. Neopentyl formate gives evidence for the cation radical, an RCH·2 radical and the fragmentation radical cation as the sample is annealed. These results show that ester cation radicals are highly reactive even at low temperatures where proton-transfer and fragmentation reactions are found.

Hydrogen abstraction reactions by amide electron adducts: A comparison to acid, ester, aldehyde and ketone electron adducts

Abstract Electron reactions with a number of peptide model compounds (amides and N-acetylamino acids) in aqueous glasses at low temperature have been investigated using ESR spectroscopy. The radicals produced by electron attachment to amides, RC(OD)NDR′, are found to act as hydrogen abstracting agents. For example, the propionamide electron adduct is found to abstract from its parent propionamide. Electron adducts of other amides investigated show similar behavior except for acetamide electron adduct which does not abstract from its parent compound, but does abstract from other amides. The tendency toward abstraction for amide electron adducts are compared to electron adducts of several carboxylic acids, ketones, aldehydes and esters. The comparison suggests the hydrogen abstraction tendency of the various deuterated electron adducts (DEAs) to be in the following order: aldehyde DEA > acid DEA ≅ ester DEA > ketone DEA > amide DEA. In basic glasses the hydrogen abstraction ability of the amide electron adducts is maintained until the concentration of base is increased sufficiently to convert the DEA to its anionic form, RC(O−)ND2. In this form the hydrogen abstracting ability of the radical is greatly diminished. Similar results were found for the ester and carboxyclic acid DEAs tested. A reinvestigation of previous work found for acetylamino acid DEA also shows evidence for hydrogen abstraction by the DEA from the parent compound. This mechanism is proposed as a likely competing reaction to the well established mechanism involving secondary deamination of the electron adduct.