Francisco A. García-Gil

Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review

Free radicals generated within subcellular compartments damage macromolecules which lead to severe structural changes and functional alterations of cellular organelles. A manifestation of free radical injury to biological membranes is the process of lipid peroxidation, an autooxidative chain reaction in which polyunsaturated fatty acids in the membrane are the substrate. There is considerable evidence that damage to polyunsaturated fatty acids tends to reduce membrane fluidity. However, adequate levels of fluidity are essential for the proper functioning of biological membranes. Thus, there is considerable interest in antioxidant molecules which are able to stabilize membranes because of their protective effects against lipid peroxidation. Melatonin is an indoleamine that modulates a wide variety of endocrine, neural and immune functions. Over the last two decades, intensive research has proven this molecule, as well as its metabolites, to possess substantial antioxidant activity. In addition to their ability to scavenge several reactive oxygen and nitrogen species, melatonin increases the activity of the glutathione redox enzymes, that is, glutathione peroxidase and reductase, as well as other antioxidant enzymes. These beneficial effects of melatonin are more significant because of its small molecular size and its amphipathic behaviour, which facilitates ease of melatonin penetration into every subcellular compartment. In the present work, we review the current information related to the beneficial effects of melatonin in maintaining the fluidity of biological membranes against free radical attack, and further, we discuss its implications for ageing and disease.

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL‐1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL‐1 solutions with or without MEL + TMZ and subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin‐1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL‐1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL‐1 alone. IGL‐1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin‐1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL‐1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.

Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury

Abstract:  Chronic organ‐donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia–reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL‐1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia–reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL‐1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia–reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well‐known cytoprotective factors as hemeoxygenase 1 (HO‐1). Fatty livers preserved in IGL‐1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL‐1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL‐1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e‐NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL‐1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia–reperfusion injury.

Melatonin Protects Against Taurolithocholic-Induced Oxidative Stress in Rat Liver

Cholestasis, encountered in a variety of clinical disorders, is characterized by intracellular accumulation of toxic bile acids in the liver. Furthermore, oxidative stress plays an important role in the pathogenesis of bile acids. Taurolithocholic acid (TLC) was revealed in previous studies as the most pro‐oxidative bile acid. Melatonin, a well‐known antioxidant, is a safe and widely used therapeutic agent. Herein, we investigated the hepatoprotective role of melatonin on lipid and protein oxidation induced by TLC alone and in combination with FeCl3 and ascorbic acid in rat liver homogenates and hepatic membranes. The lipid peroxidation products, malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA), and carbonyl levels were quantified as indices of oxidative damage to hepatic lipids and proteins, respectively. In the current study, the rise in MDA + 4‐HDA levels induced by TLC was inhibited by melatonin in a concentration‐dependent manner in both liver homogenates and in hepatic membranes. Melatonin also had protective effects against structural damage to proteins induced by TLC in membranes. These results suggest that the indoleamine melatonin may potentially act as a protective agent in the therapy of those diseases that involve bile acid toxicity. J. Cell. Biochem. 110: 1219–1225, 2010. Published 2010 Wiley‐Liss, Inc.

Potential benefits of melatonin in organ transplantation: a review

Organ transplantation is a useful therapeutic tool for patients with end-stage organ failure; however, graft rejection is a major obstacle in terms of a successful treatment. Rejection is usually a consequence of a complex immunological and nonimmunological antigen-independent cascade of events, including free radical-mediated ischemia-reperfusion injury (IRI). To reduce the frequency of this outcome, continuing improvements in the efficacy of antirejection drugs are a top priority to enhance the long-term survival of transplant recipients. Melatonin (N-acetyl-5-methoxytryptamine) is a powerful antioxidant and ant-inflammatory agent synthesized from the essential amino acid l-tryptophan; it is produced by the pineal gland as well as by many other organs including ovary, testes, bone marrow, gut, placenta, and liver. Melatonin has proven to be a potentially useful therapeutic tool in the reduction of graft rejection. Its benefits are based on its direct actions as a free radical scavenger as well as its indirect antioxidative actions in the stimulation of the cellular antioxidant defense system. Moreover, it has significant anti-inflammatory activity. Melatonin has been found to improve the beneficial effects of preservation fluids when they are enriched with the indoleamine. This article reviews the experimental evidence that melatonin is useful in reducing graft failure, especially in cardiac, bone, otolaryngology, ovarian, testicular, lung, pancreas, kidney, and liver transplantation.

Melatonin prolongs graft survival of pancreas allotransplants in pigs

Abstract:  Oxidative stress is involved in ischemia‐reperfusion injury and allograft rejection after transplantation. We studied two well‐known antioxidants, melatonin and ascorbic acid (AA), in relation to the survival of a pancreas transplantation model without immunosuppression. Forty‐eight Landrace pigs were divided into three groups (n = 16 each; eight donors and eight recipients) that received melatonin, AA, or no antioxidant therapy (controls). Melatonin and AA were administered (10 mg/kg body weight) intravenously to donors and recipients during surgery and on postoperative days 1–7. The molecules were also added (5 mm) to a University of Wisconsin preservation solution during organ cold storage. Melatonin significantly delayed acute rejection and prolonged allograft survival (25.1 ± 7.7 days) compared with the controls (8.1 ± 0.8 days, P = 0.013) and the AA group (9.4 ± 1.6 days, P = 0.049). Melatonin reduced indicators of oxidative stress, malondialdehyde, and 4‐hydroxyalkenals, in pancreatic samples collected during procurement, cold ischemia, and reperfusion. Melatonin also reduced serum pig‐major acute‐phase protein/inter‐α‐trypsin inhibitor heavy chain 4 (pMAP/ITIH4) in the early post‐transplantation period. AA only partially reduced oxidative damage 30 min postreperfusion and failed to prevent pMAP/ITIH4 elevations. These findings suggested that melatonin may be a useful therapeutic tool for organ transplantation.

Relevance of Epidermal Growth Factor to Improve Steatotic Liver Preservation in IGL-1 Solution

AIM Static preservation solution is critical for liver graft outcomes, especially when steatosis is present. Institut Georges Lopez (IGL)-1 solution protects fatty livers effectively against cold ischemia reperfusion injury. Its benefits are mediated by nitric oxide and prevention of oxidative stress. The supplementation of IGL-1 with epidermal growth factor (EGF) enhances steatotic graft preservation by increasing adenosine triphosphate content, thereby mitigating oxidative stress and mitochondrial damage. METHODS After steatotic livers were preserved for 24 hours in IGL-1 solution with or without EGF supplements, they were perfused ex vivo for 2 hours at 37°C. The benefits of EGF were assessed by evidences of hepatic damage and function--transaminases, bile production, and flow rate--as well as by other factors presumably associated with the poor tolerance of fatty livers toward cold ischemia-reperfusion injury (IRI)--energy metabolism, mitochondrial damage, oxidative stress, eNOS activity and proinflammatory interleukin (IL) beta content. RESULTS Steatotic livers preserved in IGL-1 solutions supplemented with EGF (10 μg/L) showed lower transaminase levels, greater bile production, and ameliorated flow rates when compared to IGL-1 alone. In addition, energy metabolism deterioration, mitochondrial damage, oxidative stress, and cytokine IL-1 beta release were prevented. CONCLUSION EGF addition to IGL-1 increased fatty liver graft preservation, thereby reducing steatotic liver damage against cold IRI.

Evaluation of institut georges lopez-1 preservation solution in pig pancreas transplantation: A pilot study

Background Institut Georges Lopez-1 preservation solution (IGL-1) is an emerging extracellular-type electrolyte solution, low in viscosity, containing polyethylene glycol 35 as a colloid. Although IGL-1 has shown beneficial outcomes in kidney and liver preservation, this pilot study is the first to evaluate the efficacy of IGL-1 in pancreas transplantation (PT) compared with the University of Wisconsin solution (UW). Methods Sixteen Landrace pigs underwent allogeneic PT with 16 hr of cold ischemia. Grafts were preserved with IGL-1 (n=8) or UW (n=8). No immunosuppression was administered. We analyzed graft function, the acute-phase response, and oxidative stress in the pancreatic graft monitoring membrane fluidity and lipid peroxidation. Results All eight grafts with IGL-1, but only six with UW, were functioning. Graft failures with UW resulted from graft thrombosis. There were no differences between the two solutions in the number of normoglycemic days (IGL-1: 11.5±6.2 versus UW: 8.5±4.4 days, P=0.1357), nor in lipid peroxidation during 16-hr cold ischemia (P=0.672), or reperfusion (P=0.185), but IGL-1 prevented changes in membrane fluidity after reperfusion when compared with UW (P=0.026). Conclusion IGL-1 offered the same degree of safety and effectiveness as UW in our model of pig PT with 16 hr of cold ischemia.

Inter-alpha-trypsin inhibitor heavy chain 4 as a marker of acute rejection in pancreas allotransplantation in pigs.

BACKGROUND/AIMS An early, simple, and reliable marker for acute pancreatic allograft rejection is not available. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an interleukin-6-dependent acute-phase positive protein that can act as an anti-inflammatory protein. We studied the response of the ITIH4 in pigs undergoing pancreas allotransplantation (PT) and evaluated this protein as a biomarker for acute graft rejection. METHODS PT with enteric drainage of the exocrine secretion and systemic venous drainage was performed on 12 Landrace pigs. No immunosuppression was administered. Serum concentrations of glucose, amylase, lipase, insulin, C-peptide, and ITIH4 were determined daily. RESULTS The response of ITIH4 to PT was early, intense, and prolonged, with 2 peaks in serum concentration. The first peak, which started on day 1 and reached maximum (around 6 mg/dL) on day 3, was attributed to the systemic acute phase response to surgical stress. The second peak, which exceeded the first peak and reached maximum (>8 mg/dL) on day 6, began when the recipients were still normoglycemic, and preceded onset of the diabetic state caused by acute graft rejection by an average of 4 days. CONCLUSION Serum ITIH4 could help to predict subclinical acute graft rejection after PT in pigs.

Effects of Premedication With Tiletamine/Zolazepam/Medetomidine During General Anesthesia Using Sevoflurane/Fentanyl in Swine Undergoing Pancreas Transplantation

OBJECTIVE To assess cardiac and hemodynamic responses and body temperature during long-term general anesthesia using sevoflurane/fentanyl after premedication with a tiletamine/zolazepam/medetomidine combination in swine undergoing experimental pancreas transplantation. MATERIALS AND METHODS Twelve Landrace female pigs of means weight 46.4 +/- 5.1 kg were premedicated by intramuscular administration of tiletamine/zolazepam (3.5 mg/kg), medetomidine (0.03 mg/kg), and atropine (0.02 mg/kg), before anesthesia with 0.75 minimum alveolar concentration sevoflurane and continuous intravenous fentanyl infusion (5.7 +/- 0.7 microg/kg/h). Assessment of heart rate, arterial blood pressure, and temperature in pigs undergoing allogenic pancreas transplant surgery were registered at the start of anesthesia (T0), as well as at 60 (T60), 120 (T120), and 180 (T180) minutes after T0, and finally at the end of anesthesia (T anesthesia end), when we switched off the sevoflurane vaporizer. Analysis of variance was used to determine differences between times with P < .05 considered significant. Results are given as mean values +/- standard deviations. RESULTS Arterial blood pressure significantly decreased from T120 to the end of anesthesia, while a significantly decreased heart rate was only evident at T60. Body temperature decreased significantly from T60 to the end of anesthesia. These decreases, however, lacked clinical relevance; all parameters were within normal range. No major anesthetic complications were observed in this study. CONCLUSIONS The administration of a tiletamine/zolazepam/medetomidine combination as premedication in swine subjected to pancreas transplantation allowed for a safe reduction of sevoflurane/fentanyl requirements during long-term general anesthesia. Despite arterial blood pressure and body temperature evidencing a decrease during anesthetic maintenance, all parameters remained within normal range values.