Electron paramagnetic resonance spectroscopy reveals alterations in the redox state of endogenous copper and iron complexes in photodynamic stress-induced ischemic mouse liver.

Divalent copper and iron cations have been acknowledged for their catalytic roles in physiological processes critical for homeostasis maintenance. Being redox-active, these metals act as cofactors in the enzymatic reactions of electron transfer. However, under pathophysiological conditions, owing to their high redox potentials, they may exacerbate stress-induced injury. This could be particularly hazardous to the liver - the main body reservoir of these two metals. Surprisingly, the involvement of Cu and Fe in liver pathology still remains poorly understood. Hypoxic stress in the tissue may act as a stimulus that mobilizes these ions from their hepatic stores, aggravating the systemic injury. Since ischemia poses a serious complication in liver surgery (e.g. transplantation) we aimed to reveal the status of Cu and Fe via spectroscopic analysis of mouse ischemic liver tissue. Herein, we establish a novel non-surgical model of focal liver ischemia, achieved by applying light locally when a photosensitizer is administered systemically. Photodynamic treatment results in clear-cut areas of the ischemic hepatic tissue, as confirmed by ultrasound scans, mean velocity measurements, 3D modelling of vasculature and (immuno)histological analysis. For reference, we assessed the samples collected from the animals which developed transient systemic endotoxemic stress induced by a non-lethal dose of lipopolysaccharide. The electron paramagnetic resonance (EPR) spectra recorded in situ in the liver samples reveal a dramatic increase in the level of Cu adducts solely in the ischemic tissues. In contrast, other typical free radical components of the liver EPR spectra, such as reduced Riske clusters are not detected; these differences are not followed by changes in the blood EPR spectra. Taken together, our results suggest that local ischemic stress affects paramagnetic species containing redox-active metals. Moreover, because in our model hepatic vascular flow is impaired, these effects are only local (confined to the liver) and are not propagated systemically.

Nitrosylhemoglobin in photodynamically stressed human tumors growing in nude mice.

The role of nitric oxide in human tumor biology and therapy has been the subject of extensive studies. However, there is only limited knowledge about the mechanisms of NO production and its metabolism, and about the role NO can play in modern therapeutic procedures, such as photodynamic therapy. Here, for the first time, we report the presence of nitrosylhemoglobin, a stable complex of NO, in human lung adenocarcinoma A549 tumors growing in situ in nude mice. Using electron paramagnetic resonance spectroscopy we show that the level of nitrosylhemoglobin increases in the course of photodynamic therapy and that the phenomenon is local. Even the destruction of strongly vascularized normal liver tissue did not induce the paramagnetic signal, despite bringing about tissue necrosis. We conclude that photodynamic stress substantiates NO production and blood extravasation in situ, both processes on-going even in non-treated tumors, although at a lower intensity.

Pulmonary metastases of the A549-derived lung adenocarcinoma tumors growing in nude mice. A multiple case study.

Lung adenocarcinoma is a leading human malignancy with fatal prognosis. Ninety percent of the deaths, however, are caused by metastases. The model of subcutaneous tumor xenograft in nude mice was adopted to study the growth of control and photodynamically treated tumors derived from the human A549 lung adenocarcinoma cell line. As a side-result of the primary studies, observations on the metastasis of these tumors to the murine lungs were collected, and reported in the present paper. The metastasizing primary tumors were drained by a prominent number of lymphatic vessels. The metastatic tissue revealed the morphology of well-differentiated or trans-differentiated adenocarcinoma. Further histological and histochemical analyses demonstrated the presence of golden-brown granules in the metastatic tissue, similar to these found in the tumor tissue. In contrast to the primary tumors, the electron paramagnetic resonance spectroscopy revealed no nitric oxide - hemoglobin complexes (a source of intense paramagnetic signals), in the metastases. No metastases were found in other murine organs; however, white infarctions were identified in a single liver. Taken together, the A549-derived tumors growing subcutaneously in nude mice can metastasize and grow on site in the pulmonary tissue. Thus, they can represent an alternative for the model of induced metastatic nodule formation, following intravenous administration of the cancerous cells.

Changes in the nitric oxide level in the rat liver as a response to brain injury.

Liver disturbances stimulate inflammatory reaction in the brain but little is known if injury to the brain can significantly influence liver metabolism. This problem is crucial in modern transplantology, as the condition of the donor brain seems to strongly affect the quality (viability) of the graft, which is often obtained from brain-dead donors, usually after traumatic brain injury. Because nitric oxide is one of the significant molecules in brain and liver biology, we examined if brain injury can affect NO level in the liver. Liver samples of Wistar rats were collected and studied with EPR NO-metry to detect NO level changes at different time points after brain injury. Shortly after the trauma, NO level in the liver was similar to the control. However, later there was a significant increase in the NO content in the livers starting from the 2nd day after brain injury and lasting up to the 7th day. It seems that the response to a mechanical brain injury is of the systemic, rather than local character. Therefore brain metabolism disturbances can influence liver metabolism at least by stimulating the organ to produce NO.

Changes in nitric oxide content following injury to the neonatal rat brain.

Nitric oxide is an important mediator of inflammation in the brain, but it still remains unresolved whether its action is protective or not. In particular, it seems crucial to compare the effects observed in the mature brain with the developing brain of newborn animals. The influence of NO on tissue depends significantly on its concentration. In the present study we tried to find how NO production changes after brain injury in neonatal rats. 6-day-old rats received mechanical injury to the left brain hemisphere and the tissue was collected at subsequent time points, either for EPR analysis or histochemical examination with NADPH-diaphorase staining. Our data revealed that NO concentration in the lesioned hemisphere increases slightly at 1 and 2 days after injury but also 8 days later. However, changes in the number of NADPH-diaphorase positive cells showed a different pattern from changes in NO level. These data suggest that NO concentration in the brain depends on its developmental stage.

Nitric oxide spin-trapping and NADPH-diaphorase activity in mature rat brain after injury.

Traumatic brain injury (TBI) induces inflammatory reactions, and one of the essential mediators of this reaction is nitric oxide (NO). The action of this compound is still under study because no clear consensus has been reached about its exact action in the central nervous system. Further, it is unknown if, in the damaged brain, its neuroprotective activity outweighs its putative neurodegenerative properties. Using ferrous-diethyldithiocarbamate chelate, a lipophilic spin trap for NO detection by electron paramagnetic resonance (EPR) spectroscopy, we followed NO production in injured brain of mature Wistar rats. To relate changes in the amount of NO in the lesioned brain to the activity of NO synthase (NOS), this study also used NADPH-diaphorase staining. Our data show a rapid drop of NO concentration in the damaged brain below control values. This phenomenon persisted over several hours postinjury and varied with brain region. This decrease in NO concentration was accompanied by a simultaneous increase in the number of NADPH-diaphorase-positive cells, perhaps indicative of increased NOS activity. It is therefore assumed that, in the lesioned brain, a very rapid removal of NO occurs via its transformation to other reactive species such as peroxynitrite, which further adversely influence the damaged tissue.

Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome c.

A series of multi-nanosecond molecular dynamics (MD) simulations of wild-type cytochrome c and its spin-labeled variants with the methanethiosulfonate moiety attached at position C102 were performed (1) to elucidate the effect of the spin probe presence on the protein structure and (2) to describe the structure and dynamics of the spin-label moiety. Comparisons with the reference crystal structure of cytochrome c (PDB entry: 1YCC) indicate that the protein secondary structure is well preserved during simulations of the wild-type cytochrome c but slightly changed in simulations of the cytochrome c labeled at position C102. At the time scale covered in our simulations, the spin label exhibits highly dynamical behavior. The number of observed distinct conformations of the spin label moiety is between 3 and 13. The spin probe was found to form short-lived hydrogen bonds with the protein. Temporary hydrophobic interactions between the probe and the protein were also detected. The MD simulations directly show that the disulfide bond in the tether linking a spin probe with a protein strongly influence the behavior of the nitroxide group. The conformational flexibility and interaction with the protein are different for each of the two low energy conformations of the disulfide bond.

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR.

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.