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 copper and iron 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 copper 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.