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.

High-pressure and theoretical studies reveal significant differences in the electronic structure and bonding of magnesium, zinc, and nickel ions in metalloporphyrinoids.

High pressure in combination with optical spectroscopy was used to gain insights into the interactions between Mg(2+), Zn(2+), and Ni(2+) ions and macrocyclic ligands of porphyrinoid type. In parallel, the central metal ion-macrocycle bonding was investigated using theoretical approaches. The symmetry properties of the orbitals participating in this bonding were analyzed, and pigment geometries and pressure/ligation effects were computed within DFT. Bacteriopheophytin a was applied as both a model chelator and a highly specific spectroscopic probe. The analysis of solvent and pressure effects on the spectral properties of the model Mg(2+), Zn(2+), and Ni(2+) complexes with bacteriopheophytin a shows that various chemical bonds are formed in the central pocket, depending on the valence configuration of the central metal ion. In addition, the character of this bonding depends on symmetry of the macrocyclic system. Since in most cases it is not coordinative bonding, these results challenge the conventional view of metal ion bonding in such complexes. In (labile) complexes with the main group metals, the metal ion-macrocycle interaction is mostly electrostatic. Significantly, water molecules are not preferred as a second axial ligand in such complexes, mainly due to the entropic constraints. The metal ions with a closed d shell may form (stable) complexes with the macrocycle via classical coordination bonds, engaging their p and s orbitals. Transition metals, due to the unfilled d shell, do form much more stable complexes, because of strong bonding via both coordination and covalent interactions. These conclusions are confirmed by DFT computations and theoretical considerations, which altogether provide the basis to propose a consistent and general mechanism of how the central metal ion and its interactions with the core nitrogens govern the physicochemical properties of metalloporphyrinoids.

Zinc-pheophorbide a-highly efficient low-cost photosensitizer against human adenocarcinoma in cellular and animal models.

BACKGROUND: Our previous study has shown a prolonged retention and accumulation of Zn-pheophorbide a, a water-soluble derivative of chlorophyll a, in tumor tissue (Szczygiel et al. [19]). This prompted us to further evaluate the phototherapeutic potential of this photosensitizer of excellent physicochemical properties. METHODS: Cellular uptake of Zn-pheophorbide, its localization in cells, cytotoxicity, phototoxicity and cell death mechanisms were studied in human adenocarcinoma cell lines: A549, MCF-7 and LoVo. The PDT efficacy was tested against A549 tumors growing in nude mice. RESULTS: Zn-pheophorbide a even at very low concentrations (∼1×10(-6)M) and at low light doses (5J/cm(2)) causes a strong photodynamic effect, leading to 100% cell mortality. Confocal microscopy showed that in contrast to most derivatives of chlorophyll, Zn-pheophorbide a does not localize to mitochondria. The photodynamic effects and the cell death mechanisms of Zn-pheophorbide a, its Mg analog (chlorophyllide a) and Photofrin were compared on the A549 cells. Zn-pheophorbide a showed the strongest photodynamic effect, at low dose killing all A549 cells via apoptosis and necrosis. The very high anti-cancer potential of Zn-pheophorbide was confirmed in a photodynamic treatment of the A549 tumors. They either regressed or were markedly inhibited for up to 4 months after the treatment, resulting, on average, in a 5-fold decrease in tumor volume. CONCLUSION: These results show that Zn-pheophorbide a is a very promising low-cost, synthetically easily accessible, second generation photosensitizer against human cancer.

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.

Excitation energy trapping and dissipation by Ni-substituted bacteriochlorophyll a in reconstituted LH1 complexes from Rhodospirillum rubrum.

Bacteriochlorophyll a with Ni(2+) replacing the central Mg(2+) ion was used as an ultrafast excitation energy dissipation center in reconstituted bacterial LH1 complexes. B870, a carotenoid-less LH1 complex, and B880, an LH1 complex containing spheroidene, were obtained via reconstitution from the subunits isolated from chromatophores of Rhodospirillum rubrum . Ni-substituted bacteriochlorophyll a added to the reconstitution mixture partially substituted the native pigment in both forms of LH1. The excited-state dynamics of the reconstituted LH1 complexes were probed by femtosecond pump-probe transient absorption spectroscopy in the visible and near-infrared spectral region. Spheroidene-binding B880 containing no excitation dissipation centers displayed complex dynamics in the time range of 0.1-10 ps, reflecting internal conversion and intersystem crossing in the carotenoid, exciton relaxation in BChl complement, and energy transfer from carotenoid to the latter. In B870, some aggregation-induced excitation energy quenching was present. The binding of Ni-BChl a to both B870 and B880 resulted in strong quenching of the excited states with main deexcitation lifetime of ca. 2 ps. The LH1 excited-state lifetime could be modeled with an intrinsic decay time constant in Ni-substituted bacteriochlorophyll a of 160 fs. The presence of carotenoid in LH1 did not influence the kinetics of energy trapping by Ni-BChl unless the carotenoid was directly excited, in which case the kinetics was limited by a slower carotenoid S1 to bacteriochlorophyll energy transfer.

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.

Molecular symmetry determines the mechanism of a very efficient ultrafast excitation-to-heat conversion in Ni-substituted chlorophylls.

In the Ni-substituted chlorophylls, an ultrafast (<60 fs) deactivation channel is created, which is not present in Ni-porphyrins. This observation prompted us to investigate in detail the mechanism of excitation-to-heat conversion in Ni-substituted chlorophylls, experimentally, using time-resolved laser-induced optoacoustic spectroscopy, and theoretically, using group theory approach. The Ni-substituted chlorophylls show exceptional photostability and the optoacoustic measurements confirm the prompt and very efficient (100%) excitation-into-heat conversion in these complexes. Considering their excellent spectral properties and the loss-free excitation-into-heat conversion they are likely to become a new class of versatile photocalorimetric references. The curious features of the Ni-substituted chlorophylls originate from the symmetry of a ligand field created in the central cavity. The central N-Ni(2+) bonds, formed via the donation of two electrons from each of the sp(2) orbitals of two central nitrogens to an empty [Formula: see text] hybrid centered on Ni(2+), have a considerable covalent character. The extreme rate of excited state relaxation is then not due to a ladder of the metal centered d-states, often invoked in metalloporphyrins, but seems to result from a peculiar topology of the potential energy surface (a saddle-shaped crossing) due to the covalent character of the N-Ni(2+) bonds. This is confirmed by a strong 0→0 character of electronic transitions in these complexes indicating a similarity of their equilibrium geometries in the ground (S(0)) and the excited states (both Q(X) and Q(Y)). The excitation energy is very efficiently converted into molecular vibrations and dissipated as heat, involving the central Ni(2+). These Ni-substituted pigments pose a fine exemplification of symmetry control over properties of excited states of transition metal complexes.

Structural and electronic effects in the metalation of porphyrinoids. Theory and experiment.

The structure-reactivity relationships in metalation reactions of porphyrinoids have been studied using experimental and theoretical methods. A series of eight porphyrinoic ligands, derivatives of chlorophylls, was prepared in which both the peripheral groups and the degrees of saturation of the macrocycle were systematically varied. To reveal the solvent and structural factors which control the interactions of these macroligands with metal centers, their interactions with reactive Zn(2+) and inert Pt(2+) ions were investigated using absorption spectroscopy. In parallel, quantum chemical calculations (density functional theory, DFT) were performed for the same set of molecules to examine the influence of structural and electronic factors on the energy of the frontier orbitals, the nucleophilicity/electronegativity of the macrocycle, its hardness, and conformation. These static descriptors of chemical reactivity, relevant to metalation reactions, were verified against the results obtained in the experimental model. The experimentally obtained kinetic data clearly show that the solvent has a crucial role in the activation of the incoming metal center. In terms of chelator structure, the largest effects concern the size of the delocalized pi-electron system and the presence of side groups. Both the DFT calculations and experimental results show the strong influence of the macrocycle rigidity and of the peripheral groups on the chelating ability of porphyrinoids. In particular, the peripheral functionalization of the macrocyclic system seems to drastically reduce its reactivity toward metal ions. The effect of peripheral groups is two-fold: (i) a lower electron density on the core nitrogens, and (ii) increased rigidity of the macrocycle. The outcomes of the theoretical and experimental analyses are discussed also in terms of their relevance to the mechanism of biological metal insertion in the biosynthesis of heme and chlorophyll.