Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

New approaches for study of mechanisms of bone demineralization due to microgravity

Changes in nano-scale subsystems of rat femurs due to the axial unloading of hindlimbs are studied by means of electron paramagnetic resonance (EPR). After irradiation by 60Co isotopes, the results indicate that weightlessness simulation leads to formation of free radicals in tropocollagen molecules and to a reduction in the amount of CO2 radicals, located on the surface of bioapatite nanocrystals.     

Carbon dioxide stimulates the production of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite

Reaction of peroxynitrite with the biological ubiquitous CO(2) produces about 35% yields of two relatively strong one-electron oxidants, CO(3) and ( small middle dot)NO(2), but the remaining of peroxynitrite is isomerized to the innocuous nitrate. Partial oxidant deactivation may confound interpretation of the effects of HCO3-/CO(2) on the oxidation of targets that react with peroxynitrite by both one- and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that HCO3-/CO(2) partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the effects of HCO3-/CO(2) on two- and one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that HCO3-/CO(2) diverts thiol oxidation by peroxynitrite from two- to one-electron mechanisms particularly at neutral pH. At acid pH values, thiol oxidation to free radicals predominates even in the absence of HCO3-/CO(2). In addition to the previously characterized thiyl radicals (RS.), we also characterized radicals derived from them such as the corresponding sulfinyl (RSO.) and disulfide anion radical (RSSR.-) of both GSH and cysteine. Thiyl, RSO. and RSSR.- are reactive radicals that may contribute to the biodamaging and bioregulatory actions of peroxynitrite.     

Ascorbic acid radicals induced by the action of radiation in tissues from rat organs frozen at 77 K

Subsequent annealing technique and computer assistant analysis of EPR spectra were used to isolate an asymmetric EPR signal Rs(g = 2,0051; delta H = 0.8 mT) from the EPR spectrum of rat spleen gamma-irradiated at 77 K. Radicals with the same EPR spectrum were registered in: 1) water solution of ascorbic acid (2.10(-2) M, pH 3.4) frozen and irradiated at 77 K and 2) water-glycerol solution of ascorbic acid (10(-2) M, pH 10.3) frozen rapidly at the moment of intensive autooxidation. These model experiments allow to conclude that Rs signal is caused by the radicals of semidehydroascorbic acid. Radiochemical yield of these radicals as well as of all the radicals induced by gamma radiation in the whole rat tissues were measured. The EPR signal (Rs) is equivalent to the well known "artifact" signal of lyophilized tissues. The explanation of the mechanism of the radicals formation taking place under annealing of the frozen and irradiated tissues was suggested.     

X- and Q-band electron paramagnetic resonance of CO2- in hydroxyapatite single crystals

Both X- and Q-band electron paramagnetic resonance (EPR) research has been conducted using slightly carbonated hydroxyapatite (HAp) single crystals after exposure to ionizing radiation. Below a temperature of 90 K, O(-) and CO(2-) radicals were detected, whereas at room temperature only CO(2-) spectra could be observed. The O(-) ion has previously been investigated in high-purity HAp single crystals, whereas EPR spectra of CO(2-) in HAp single crystals have not been reported. Both paramagnetic defects exhibit EPR angular variations in planes containing the c axis of the crystal from which spin Hamiltonian parameters were derived. Arguments are given for the presence of two CO(2-) defects in the irradiated HAp single crystals.     

Mechanisms of gravity-dependent changes in the bone tissue

The most typical changes for the bone under the space flight conditions and a long-term hypokinesia are the following: the decreasing in bone mass, the demineralization and a reducing of a mechanical strength. It can lead to osteopenia and osteoporosis development. Also it increases the risk of fractures of supporting bones. Osteopenies, caused by the microgravity, are partially connected with the increasing of a reduction of trabecular bones. [Cytological mechanisms of gravity-dependent reactions in a bone tissue remain in many respects not clear. The study purpose was the analysis of some ultrastructural changes in bone tissue cells of the monkeys (Macaca mulatta), staying during 2 weeks onboard the biosatellite "Bion-11".     

Mechanism of polyene antibiotic inactivation. Changes in the physicochemical characteristics and the destruction of the polyene chromophore levorin during inactivation

The results of studying free radicals of some polyenic antibiotics with the EPR method are presented. It is shown that the number of free radicals increased by 100 per cent with a 2-fold decrease in the biological activity. A qualitative change in the EPR spectrum due to the presence of a new radical type was also observed. The changes in the spectral characteristics of levorin allowed one to demonstrate that breaking of the links and formation of the polymer products during oxidative destruction of the polyenic chromophore resulted from attachment of the radicals through the antibiotic double bonds. The data correlate with the results of the quantum-chemical evaluation of the polyenic chromophore.     

EPR spectral investigation of radiation-induced radicals of gallic acid

In the present work, spectroscopic features of the radiation-induced radicals of gallic acid compounds were investigated using electron paramagnetic resonance (EPR) spectroscopy. While un-irradiated samples presented no EPR signal, irradiated samples exhibited an EPR spectrum consisting of an intense resonance line at the center and weak lines on both sides. Detailed microwave saturation investigations were carried out to determine the origin of the experimental EPR lines. It is concluded that the two side lines of the triplet satellite originate from forbidden “spin-flip” transitions. The spectroscopic and structural features of the radiation-induced radicals were determined using EPR spectrum fittings. The experimental EPR spectra of the two gallic acid compounds were consistent with the calculated EPR spectroscopic features of the proposed radicals. It is concluded that the most probable radicals are the cyclohexadienyl-type, \({\dot{\text{O}}}\left( {\text{OH}} \right)_{ 2} {\text{C}}_{ 6} {\text{H}}_{ 2} {\text{COOH}}\) radicals for both compounds.     

Histomorphometric study of tibia of rats exposed aboard American Spacelab Life Sciences 2 Shuttle Mission

Tibial bones of rats flown onboard the SLS-2 shuttle mission were studied. Trabecular bone parameters were investigated, including growth plate height, trabecular bone volume, thickness and number, and trabecular separation in the primary and secondary spongiosa. Several histomorphometric changes were noted, allowing researchers to conclude that exposure to microgravity resulted in osteopenia of spongy bone of tibial metaphysis. The roles of bone formation and bone resorption are discussed.     

Multifrequency EPR study of carbonate- and sulfate-derived radicals produced by radiation in shells and corallite

Shells of two sea mollusks (Venus sp.), pearl oyster (Meleagrina vulgaris) and corallite (white coral) were exposed to ionizing radiation (gamma and X rays) and then examined by EPR spectroscopy in X, Q and W band. The resulting spectra were analyzed and the g values of the EPR lines in the multicomponent spectra were determined. The increased resolution in Q- and W-band spectra allowed us to assign the observed lines to CO(2)(-) ion radicals (isotropic and orthorhombic), SO(2)(-) isotropic, SO(3)(-) (isotropic and axial), and Mn(2+) species. The assignments were confirmed by simulations of the spectra. Practical implications for the use of Q and/or W band in low-dose quantitative EPR measurements for dating and for accidental dose estimation are discussed.     

Changes in bone mineral density and microarchitecture in cosmonauts after a six-month space flight

The effect of microgravity on the bone tissue of cosmonauts has been studied after a six-month space flight. The volumetric bone mineral density (VBMD) and the bone structural characteristics of distal segments in the radius and tibia have been studied by means of peripheral quantitative computed tomography (pQCT). The changes in VBMD were found to correlate with the position of the bone relative to the vector of gravity. In the radius, reversible hypermineralization, together with thickening of the compact bone were recorded. In the tibia, reversible osteopenia was characterized by significant losses in both compact and trabecular bones. Irrespective of the position relative to the vector of gravity, there was a trend towards microarchitectural deterioration, such as a decrease in the trabecula number and increase in the bone tissue heterogeneity. Postflight dynamics of structural parameters showed an integrative character with nonlinear time dependence.     

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [S?rlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.     

Validation of the doubly labeled water method in rats during isolation and simulated weightlessness

Total energy expenditure (TEE) of rats during simulated microgravity is unknown. The doubly labeled water method (DLW) reliably measures TEE, but the results depend on the methods of calculation. These methods were validated and appraised by indirect calorimetry in eight rats during isolation (7 days) and simulated microgravity (10 days). There were no effects on CO(2) production in the method used to derive constant flux rates as in the regression models. r(CO(2)) estimates were dependent on the assumed fractionation processes, the derivation of constant flux rate methods, and the selected pool models. Use of respiratory or food quotients did not influence TEE estimations, which were similar during isolation and simulation. During either isolation with growth or simulation with a stabilized mass, the one-pool model of Speakman (Speakman JR. Doubly Labelled Water. Theory and Practice. London: Chapman and Hall, 1997) resulted in the more reliable validation (0.8 +/- 2.2 and 2.2 +/- 3.4% vs. calorimetry, respectively). However, during simulation, agreement was also observed with the single pool model of Lifson (Lifson N, Gordon GB, and McClintock R. J Appl Physiol 7: 704-710, 1955) (-2.5 +/- 2.5%), and two two-pool models [Schoeller (Schoeller DA. J Nutr 118: 1278-1289, 1988) (0.5 +/- 3.1%) and Speakman (Speakman, JR. Doubly Labelled Water. Theory and Practice. London: Chapman and Hall, 1997) (-1.9 +/- 2.7%)]. This latter finding seems linked to the stable body mass and to fractionation consideration close to the single-pool model of Speakman. During isolation or simulated microgravity, the other equations underestimated TEE by 10-20%.     

Modification of the structure of plasmatic membranes of the liver by the action of alpha-tocopherol in vitro

The effect of the natural antioxidant alpha-tocopherol in a broad concentration range (10(-4) - 10(-25) M) on the viscosity characteristics and thermally induced structural transitions of a lipid bilayer of plasma membranes of murine hepatocytes in vitro has been studied. Changes in the rigidity of surface (approximately Abb) of the lipid bilayer were measured on a Bruker EMX EPR spectrometer (Germany) by the method of spin probes. Stable nitroxyl radicals of 5- and 16-doxylstearic acid, localized at different depth in the membrane served as spin probes. It was shown that the concentration dependence of the effect of alpha-tocopherol is linear and polymodal with three statistically significant increases in three ranges of its concentration: (1) in the range of traditional physiological concentrations 10(-4)-10(-9) M, (2) in the range of superlow doses 10(-9) - 10(-17) M, and (3) in the range of "imaginary" concentrations 10(-17) - 10(-25) M. The mechanisms of action of alpha-tocopherol in each of the three ranges are discussed. When studying the temperature dependences of viscous characteristics, a new thermally induced structural transition in the range of "physiological" temperatures 309-313 K for those alpha-tocopherol concentrations (including superlow ones) to which the maxima on the dose dependence curves at constant temperature of 293 K corresponded.     

Structural studies of the biomineralized species of calcified pancreatic stones in patients suffering from chronic pancreatitis

The pancreatic stones (Pancreatic calculi) collected from patients suffering from chronic calcific pancreatitis were studied in a view to explore the constituents involved in the calcification. The calcified stones were characterized by XRD, EPR and IR spectroscopic techniques. The detailed studies indicate that these stones consist of calcium carbonate as a major component, besides phosphates and other protein content. The presence of aragonite phases in the biomineralized stones is also discussed. The EPR spectra gave an evidence of the presence of traces of manganese in different oxidation states, which is used as one of the EPR probes in the present work. The samples were sintered at different temperatures to remove all the organic matter, and their EPR spectra have been studied to obtain detailed information regarding the changes in the symmetry of these stone samples. The X-irradiated sample was also characterized by EPR and the resonance signals are attributed to freely rotating CO(2)(-) radicals. The infrared spectrum reveals the presence of many organic bands corresponding to the protein amides.     

The effects of ionizing radiation on DNA: the role of thiols as radioprotectors

Electron loss from N-(2-mercaptopropionyl) glycine (PSH) gave an EPR detectable radical anion, PS-.SP(-). When the PSH derivative was frozen in aqueous DNA solutions to 77 K and exposed to ionizing radiation, normal damage to the DNA was detected by EPR spectroscopy. However, on annealing above 77 K, central EPR features for the DNA base radical cations and anions gave central features assigned to PS-.SP(-) sigma*-radical anions, together with outer features for 5-6-dihydro-5-thymyl radicals, TH.. It is proposed that on freezing, the PSH molecules are constrained into a glassy region around the DNA, and that, on annealing, electron donation gives PS. radicals, with loss of quanine radical-cations, G(.+). The PS. radicals were not detectable, but on reaction with another PSH molecule, gave good EPR spectra for PS-.SP(-) radical-anions. These results indicate that PSH had little effect on the yield of the other base radicals C(.-)/T(.-). Also, growth of TH. radicals, formed from protonated thymine radical-anions, T(.-), were detected. We conclude that the primary effect of PSH is to capture the G(.+) centers, and thus could either prevent or repair radiation damage to DNA.     

Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts

Astronauts experiencing long periods of space flight suffer from severe loss of bone tissue, particularly in those bones that carry the body weight under normal gravity. It is assumed that the lack of mechanical load decreases connective tissue biosynthesis in bone-forming cells. To test this assumption, quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity, and hypergravity conditions were investigated by incubating human fibroblast cultures with [³H]-proline for 4, 7, 10, and 20 h during the Spacelab D2-mission in 1993. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being up to 143% higher than in 1 g controls. In contrast, hypergravity samples showed a decrease in collagen synthesis with increasing g, being at the 13% level at 10 g. The relative proportion of collagen in total synthesized protein showed a slight decrease with increasing g. The secretion of collagen by the cells, proline hydroxylation of individual collagen -chains, and the relative proportions of synthesized collagens I, III, and V were not affected under any of the applied conditions.Our research was supported financially by Dara GmbH Bonn (grant. no. 01QV 8866), the Deutsche Forschungsgemeinschaft (SFB A1/367) and BMFT grant. no. 01 KM 9303/8.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

Spin Trapping of Methyl Radicals by the Acyl Nitroso Compound PH-C (= O)NO Formed in the Photochemical Reaction Between Benzohydroxamic Acid, Dimethyl Sulfoxide and Hydrogen Peroxide. An Epr Study

By the use of EPR spectroscopy, it has been shown that acyl nitroso compounds can act as spin traps for short-lived radicals with the formation of acyl aminoxyl radicals. The reaction was studied for the system benzohydroxamicacid[Ph-C (= O)N(H)] - dimethyl sulfoxide - hydrogen peroxide. The acyl aminoxyl radicals appeared almost immediately when the reaction mixture was irradiated in situ in the EPR cavity with UV light. The trapping reaction involved two photochemical reactions, i.e. the oxidation of the hydroxamic acid to the acyl nitroso compound Ph-C (= O)NO, and the formation of methyl radicals from dimethyl sulfoxide. The EPR spectra are superpositions of the spectra of two species of acyl aminoxyl radicals, i.e. the radicals Ph-C (= O)N(O·)H formed by oxidation of the parent benzohydrox-amic acid, and the radical Ph-C (= O)N(O·)CH₃, formed by trapping of methyl radicals.