Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

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.

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.

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.

Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR.

Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO. We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia. We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress.