The effect of a synthetic hexadentate iron chelator (CP130) and desferrioxamine on rabbit kidneys exposed to cold and warm ischaemia.

The effect of CP130 (a synthetic hexadentate pyridinone iron chelator) on the formation of two markers of lipid peroxidation (TBA-reactive material and Schiff's bases) in rabbit kidneys following a 72 h period of cold (0-4 degrees C) ischaemia was investigated by either adding CP130 to the flush/storage solution (hypertonic citrate solution) or by administering the agent intravenously 15 min before removal of the organs. In both cases, CP130 blocked the adverse rises in lipid peroxidation caused by ischaemia and subsequent reoxygenation of the homogenates in vitro. Both CP130 and desferrioxamine (DFX) (administered intravenously 15 min before ischaemia and 5 min before reperfusion) were also found to significantly reduce post-ischaemic rates of in vitro lipid peroxidation in kidneys rendered warm ischaemic for 90 min followed by reperfusion for 5 or 60 min in situ. Kidneys exposed to warm ischaemia and reperfusion developed interstitial and intracellular oedema, congestion and haemorrhage. DFX administration had little effect on the histological outcome, whereas CP130 significantly reduced interstitial oedema (at 5 min reperfusion compared to the DFX-treated group), intracellular oedema (at 60 min reperfusion compared to the DFX-treated group) and congestion (at 5 min reperfusion compared with a control group not given any agent). It is concluded that while CP130 and DFX exhibited similar antioxidant properties, CP130 provided better protection from ischaemia/reperfusion injury at the histological level. Synthetic iron chelators may therefore be of benefit in clinical organ transplantation by protecting against tissue damage caused by prolonged ischaemia.

Reoxygenation following hypoxia stimulates lipid peroxidation and phosphatidylinositol breakdown in kidney cortical slices.

Reoxygenation of hypoxic (120 min at 37 degrees) rabbit kidney cortical slices in vitro resulted in a rapid increase in lipid peroxidation and phosphatidylinositol hydrolysis. No changes in phosphatidylinositol breakdown occurred during hypoxia or upon reoxygenation in the absence of calcium. Incubation of renal slices with carbon tetrachloride resulted in increased lipid peroxidation but had no effect on phosphatidylinositol breakdown. It is concluded that altered intracellular calcium homeostasis during reoxygenation is involved in mediating increased phosphatidylinositol hydrolysis through activation of a specific phospholipase C, but that oxidative stress per se does not have a significant effect on the inositol phosphate secondary messenger response in this model system.

Ultrastructural changes and lipid peroxidation in rat adipomusculocutaneous flap isotransplants after normothermic storage and reperfusion.

Parallel in vivo, histological, and ultrastructural studies were carried out and markers of lipid peroxidation (Schiff's bases [SB] and thiobarbituric-acid-reactive material [TBAR]) were measured in rat adipomusculocutaneous flap isotransplants that had been stored for 0, 2, 4, 6, and 8 hr under normothermic (37 degrees C) conditions and reperfused for specific periods. Flaps stored for 4 hr and treated with intravenous desferrioxamine (DFX) or hypertonic citrate flush (HCA) were also evaluated. In vivo assessment was made after 7 days of reperfusion. Flaps stored for 4 hr eventually exhibited partial necrosis in vivo, and neither DFX or HCA flush increased the area of surviving skin. Electron microscopy revealed extensive storage damage in epidermal, follicle, fat, and smooth muscle cells and in endothelium. HCA significantly preserved fat cells (P = 0.0035) and DFX diminished smooth muscle damage. Reperfusion injury was seen in endothelial cells in the form of swelling that was not prevented by HCA or DFX. Ultrastructural alterations correlated with changes in susceptibility to lipid peroxidation in fat but not in skin. The results of these parallel studies indicate that both free radical-dependent and independent mechanisms operate in ischemia and reperfusion injury in flap tissue and that fat has a greater predisposition to free radical damage than skin.

Ebselen. Antioxidant capacity in renal preservation.

Ebselen (PZ51) was tested for its ability to inhibit oxidative membrane damage and improve outcome of rabbit kidneys rendered cold ischaemic for 72 hr. In view of the rapid metabolism of ebselen, the antioxidant capacities of its two principal metabolites were first compared with that of the parent drug in an in vitro hepatic microsomal lipid peroxidation system initiated by NADPH/Fe(3+)-ADP. The potent antioxidant activity of ebselen was confirmed but metabolite I (2-glucuronylselenobenzanilide) exhibited no antioxidant potential up to a concentration of 50 microM; metabolite II (4-hydroxy-2-methyl-selenobenzanilide) did inhibit lipid peroxidation but was about 80 times less effective than the parent compound. The storage of rabbit kidneys in hypertonic citrate solution at 0 degrees for 72 hr of cold ischaemia resulted in greatly increased susceptibility to oxidative membrane damage in both the cortex and medulla as determined by the subsequent in vitro formation of two markers of lipid peroxidation (Schiff's bases and thiobarbituric acid-reactive material). Inclusion of ebselen (50 microM) in the flush and storage solution led to a highly significant reduction in these oxidative markers in both regions of the kidney. Intracellular and interstitial oedema was noted in organs subjected to 72 hr cold ischaemia and was reduced by ebselen (50 microM in the flush/storage solution). The rate of post-ischaemic lipid peroxidation was found to correlate well with the extent of oedema in the renal medulla (r = 0.84, P less than 0.001) but no such correlation was found in the cortex. Administration of ebselen (5.5 mg/kg i.v. and 100 microM in the flush/storage solution) did not improve the long-term survival of rabbits following autotransplantation of a single kidney stored for 48 or 72 hr. No protective effect of ebselen could be demonstrated either in terms of graded physiological function or histological outcome.

A chelator is required for microsomal lipid peroxidation following reductive ferritin-iron mobilisation.

In the past, antioxidant and chelator studies have implicated a role for iron-dependent oxidative damage in tissues subjected to ischaemia followed by reperfusion. As ferritin is a major source of iron in non-muscular organs and therefore a potential source of the iron required for oxygen radical chemistry, we have determined conditions under which ferritin iron reduction leads to the formation of a pool of iron which is capable of catalysing lipid peroxidation. Under anaerobic conditions and in the presence of rat liver microsomes, flavin mononucleotide (FMN) catalysed the reduction of ferritin iron as shown by both continuous spectrophotometric measurements of tris ferrozine-Fe(II) complex formation and post-reaction Fe(II) determination. The presence of either ferrozine or citrate was not found to alter the time course or extent of ferritin reduction. In contrast, the addition of air to the reactants after a 20 min period of anaerobic reduction resulted in peroxidation of the microsome suspension (as determined with the 2-thiobarbituric acid test) only in the presence of a chelator such as citrate, ADP or nitrilotriacetic acid. These results support the concept that reduced ferritin iron can mediate oxidative damage during reperfusion of previously ischaemic tissues, provided that chelating agents such as citrate or ADP are present.

Function of single rat lung isografts after 48-hour cold storage. The effect of treatment with free radical antagonists and prostacyclin PGI2.

Single orthotopic rat lung isografts were carried out in adult male AS rats after 48-hour cold storage (0 degrees C). Grafts were preserved by simple organ flush followed by low-temperature immersion. Hypertonic citrate (HCA) without additives was evaluated as the basic flush solution. In other groups desferrioxamine (an iron chelator), verapamil (a calcium channel blocker) and prostacyclin (PGI2) were added separately to HCA and given intravenously to donor and recipient animals in an attempt to improve the preservation. Baseline controls were fresh HCA-flushed lungs grafted immediately after harvest. Negative controls to the HCA assessment were lungs flushed with isotonic saline (NaCl) stored for 48 hr at 0 degrees C. Functional studies were carried out at weekly intervals until sacrifice (in the fifth postoperative week) and included assessment of blood flow, aeration and gas transfer by perfusion scintigraphy, chest roentgenograms, and blood gas analysis. Of the baseline control animals, 10/10 survived to the end of the study period; all grafts appeared macroscopically normal and blood gas analysis showed good function. Of the animals grafted with HCA-flushed, 48-hr-stored lungs 2/10 died postoperatively; 7/10 grafts appeared macroscopically normal at the end of the study, and one was slightly reduced in size. Blood gas analysis of HCA-flushed, 48-hr-stored lungs showed function similar to that of baseline control grafts. NaCl-flushed lungs (negative controls) survived surprisingly well: 3/10 animals died postoperatively, 6/10 lungs appeared normal, and one was reduced in size. Assessment of graft function showed no significant benefit of HCA flush compared with NaCl. Treatment with desferrioxamine, verapamil or prostacyclin (PGI2) failed to improve the outcome after HCA flush; in fact desferrioxamine gave significantly poorer results. The study has shown that successful 48-hr preservation of rat lung isografts can be achieved by simple organ flush with HCA and storage at 0 degrees C. Contrary to expectation and experience with preservation of other organs, rat lungs were remarkably well preserved after flush with NaCl.

Lipid peroxidation and ultrastructural changes in rat lung isografts after single-passage organ flush and 48-hour cold storage with and without one-hour reperfusion in vivo.

Rat lung isografts were preserved for 48 hr at 0 degrees C using a simple organ flush technique. After storage alone, isotonic saline flush resulted in significantly raised indices of lipid peroxidation in vitro (Schiff bases and thiobarbituric-acid-reactive material [TBAR]). Lungs flushed with hypertonic citrate (HCA) had significantly less oxidative damage than saline-flushed lungs. The addition to the HCA flush of verapamil, a calcium channel blocker, or desferrioxamine, an iron chelator, significantly reduced TBA reactivity in stored lungs compared with HCA alone. After 1-hr reperfusion in vivo, lipid peroxidation was reduced in HCA-flushed lungs compared with saline flush (TBAR alone), but no additional protection from the use of desferrioxamine or verapamil was demonstrated. Electron microscopy after saline flush and storage alone showed gross endothelial swelling and fragmentation. Reperfusion with blood for 1 hr resolved cell swelling, but alveolar/capillary wall rupture occurred. HCA protected against cell swelling, but endothelial vesiculation and widening of the basement membrane were observed. After reperfusion, HCA-flushed lungs developed much endothelial loss that was considerably reduced by the use of desferrioxamine and verapamil. The lipid peroxidation results suggest that iron- and calcium-mediated free radical production may be important mechanisms in oxidative damage to stored rat lungs. Electron microscopy findings correlated with biochemical evidence of free-radical-mediated injury. Reduction of endothelial loss on reperfusion by the use of verapamil and desferrioxamine provides circumstantial evidence that ischemia and reperfusion damage of organs stored for transplantation is partly due to Fe++(+)- and Ca+(+)-dependent mechanisms that probably involve increased free radical production.

Protection against endotoxin-induced foetal resorption in mice by desferrioxamine and ebselen.

Endotoxin was administered to mice on their 13th day of pregnancy at doses which caused the resorption of approximately 50% of the implanted foetuses. The iron chelator desferrioxamine was found to significantly inhibit the percentage of resorptions induced by endotoxin in a dose-dependent manner. The highest dose of desferrioxamine (5 mg) given intravenously 30 min prior to, immediately after, and 4 and 24 h after endotoxin inoculation, reduced the percentage of resorptions from 56.9 to 17.9%. Administration of the novel selenium-containing compound ebselen, which is both an antioxidant and an inhibitor of leukotriene synthesis, was also found to significantly protect against endotoxin-induced foetal resorptions, reducing the percentage of resorbed foetuses from 52.9 to 26.0% when given at a dose of 50 mg/kg (s.c.) at the time of endotoxin inoculation and 24 and 48 h following. Both these compounds also significantly reduced the increase in spleen weights observed when the mice were given endotoxin. These results provide evidence that the iron-catalysed production of hydroxyl radicals from other oxygen-derived species and the formation of leukotrienes play an important role in the mechanism by which endotoxin causes foetal resorptions in the mouse.

Oxidative damage to kidney membranes during cold ischemia. Evidence of a role for calcium.

Storage of rabbit kidneys at 0 degrees C for periods of 72 hr after flushing with hypertonic citrate solution, or 24 hr when flushed with isotonic saline, resulted in significant increases in Schiff base and thiobarbituric acid-reactive markers of lipid peroxidation in vitro. The extent of lipid peroxidation was not significantly altered by addition of verapamil (100 microM), a Ca++ channel blocking agent, or calcium 1 mM (CaCl2) to the HCA storage solution. In contrast, verapamil significantly reduced the extent of lipid peroxidation in kidneys stored in saline solution, and a significant increase in oxidative damage occurred when CaCl2 was added to this storage solution. Thus the extent of lipid peroxidation in kidneys stored in saline was significantly mediated by extracellular Ca++, whereas in HCA this was probably chelated by the large excess of citrate (55 mM) in this medium that prevented, or at least slowed, its entry into the renal cells. Lipid peroxidation was however significantly increased in kidneys stored in both HCA and saline solutions by addition of the calcium ionophore A23187 (10 microM) or the polysaccharide dye ruthenium red (5 microM) that inhibits mitochondrial uptake of Ca++. This strongly suggested that altered intracellular Ca++ homeostasis during the storage period played an important role in the development of oxidative damage to kidneys stored in both these media.

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin.

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiff's base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiff's bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiff's bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.

Determination of desferrioxamine-available iron in biological tissues by high-pressure liquid chromatography.

Intracellular iron loosely bound to proteins such as ferritin or in the form of low molecular weight chelates is available to catalyze adverse reactions such as the formation of reactive free radicals. A method to measure this small but important iron pool by utilizing the highly specific iron-chelator desferrioxamine is described. Following incubation of tissue fractions with desferrioxamine, the parent compound and its iron-bound form, ferrioxamine, are extracted using solid-phase cartridges and quantitated by reversed-phase HPLC using uv detection. Calculation of the ferrioxamine:desferrioxamine ratio and comparison with a standard curve constructed using a series of known iron concentrations allow the determination of micromolar amounts of desferrioxamine-available iron in biological samples.

The importance of iron, calcium and free radicals in reperfusion injury: an overview of studies in ischaemic rabbit kidneys.

An overview of a series of experiments attempting to link iron and calcium redistribution and release of free fatty acids with falls in pH and adenine nucleotide levels during cold storage of rabbit kidneys is presented. The data reviewed strongly suggest that these events are inextricably linked to subsequent reperfusion injury. Circumstantial evidence incriminating iron was provided by experiments showing that iron chelation decreased reperfusion injury after warm (WI) and cold ischaemia (CI) in rat skin flap and rabbit kidney models. Evidence for a role for calcium was provided when it was found that a calcium channel blocking agent added to the saline flush solution before storage inhibited lipid peroxidation, whereas chemicals which caused release or influx of calcium into the cell exacerbated oxidative damage. Additional involvement of breakdown products of adenine nucleotides was suggested by the protection from lipid peroxidation afforded by allopurinol. Involvement of calcium-activated phospholipase A2 was strongly suggested by increases in free fatty acids during cold storage and both this increase and lipid peroxidation were inhibited by addition of dibucaine to the storage solution.

A role for dietary lipids and antioxidants in the activation of carcinogens.

The ways in which dietary polyunsaturated fats and antioxidants affect the balance between activation and detoxification of environmental precarcinogens is discussed, with particular reference to the polycyclic aromatic hydrocarbon benzo(a)pyrene. The structure and composition of membranes and their susceptibility to peroxidation is dependent on the polyunsaturated fatty acid (PUFA) content of the cell and its antioxidant status, both of which are determined to a large degree by dietary intake of these compounds. An increase in the PUFA content of membranes stimulates the oxidation of precarcinogens to reactive intermediates by affecting the configuration and induction of membrane-bound enzymes (e.g., the mixed-function oxidase system and epoxide hydratase); providing increased availability of substrates (hydroperoxides) for peroxidases that cooxidise carcinogens (e.g., prostaglandin synthetase and P-450 peroxidase); and increasing the likelihood of direct activation reactions between peroxyl radicals and precarcinogens. Antioxidants, on the other hand, protect against lipid peroxidation, scavenge oxygen-derived free radicals and reactive carcinogenic species. In addition some synthetic antioxidants exert specific effects on enzymes, which results in increased detoxification and reduced rates of activation. The balance between dietary polyunsaturated fats, antioxidants and the initiation of carcinogenesis is discussed in relation to animal models of chemical carcinogenesis and the epidemiology of human cancer.

The oxidation of benzo[a]pyrene-7,8-dihydrodiol mediated by lipid peroxidation in the rat intestine and the effect of dietary lipids.

This study has demonstrated that the microsomal fraction of the rat small intestinal mucosa has the capacity to catalyse the oxidation of benzo[a]pyrene(BP)-7,8-diol to BP-diol-epoxides (BPDEs) both by a mechanism involving the mixed-function oxidase system (NADPH-dependent) and as a result of the initiation of peroxidation of the membrane phospholipids by ferrous ions, ascorbate and ADP. The NADPH-dependent reaction was fastest in the proximal part of the intestine and resulted in the formation of approximately equal amounts of BPDE I and BPDE II. The lipid peroxidation-catalysed reaction favoured the production of BPDE I and was maximal in the middle region of the intestine, closely paralleling the rate of lipid peroxidation in the intestinal sections. Feeding rats on a cod liver oil diet, rich in C20:5 and C22:6, significantly increased the incorporation of these fatty acids into the microsomal fractions. This resulted in a greatly increased rate of lipid peroxidation in vitro and a significantly higher rate of lipid peroxidation-catalysed BP-7,8-diol oxidation compared to rats fed fat-free, mono-unsaturated lard or corn oil (58% C18:2) diets. Thus the rate of conversion of BP-7,8-diol to its ultimate carcinogenic forms during lipid peroxidation in the intestinal fractions of rats fed a polyunsaturated fat was quantitatively more important than the NADPH-catalysed reaction as measured in vitro.

The effect of dietary lipids and antioxidants on the activity of epoxide hydratase in the rat liver and intestine.

The effect of varying the fatty acid composition of the lipid components of the diet on the activity of epoxide hydratase in the rat liver and intestinal mucosa has been studied. Feeding a 10% cod liver oil diet (containing 18% C20:5 and 11% C22:6) resulted in a 3-fold increase in epoxide hydratase activity in the liver and a 1.6-fold increase in the intestine compared to rats fed a fat-free diet. The activity of epoxide hydratase in rats fed a cod liver oil diet was significantly greater than that for the group fed a lard diet (containing mainly saturated and mono-unsaturated fatty acids) containing the same quantity of vitamin E. Thus, the enhancing effect of the cod liver oil diet was due to the polyunsaturated fatty acids in this oil. Dietary corn oil (58% C18:2) also stimulated epoxide hydratase activity in the liver but not in the intestine. Vitamin E levels of up to 500 mg/kg diet were ineffective at inducing epoxide hydratase activity in both the liver and intestine. Significant changes in the fatty acid composition of hepatic and intestinal microsomes took place when rats were fed diets of different fatty acid composition. These changes were such that the proportions of polyunsaturated fatty acids in the microsomal fractions reflected the amounts of these fatty acids in the dietary fat. Hepatic epoxide hydratase activity was found to be positively correlated to the proportion of polyunsaturated fatty acids in the microsomal fractions of the liver.

The oxidation of benzo[a]pyrene mediated by lipid peroxidation in irradiated synthetic diets.

The effect of gamma-irradiation (1000-4000 Gy) on the formation of lipid peroxides and on the oxidation of the environmental carcinogen benzo[a]pyrene (BP) has been studied in mixtures of starch/fat and BP which were used as models for natural foods. When mixtures containing polyunsaturated fats (mackerel oil and cod-liver oil which contain relatively large proportions of C20:5 and C22:6) were exposed to gamma-irradiation, large concentrations of lipid peroxide were formed and a concomitant oxidation of BP to mutagenic and toxic BP quinones took place. The rate of BP oxidation was closely related to the extent of peroxidation of the lipids in the starch mixtures and was dependent on the dose of gamma-irradiation and the presence of air. Mackerel oil also underwent peroxidation during the storage of both irradiated and unirradiated starch/mackerel oil/BP mixtures and this resulted in a significant oxidation of the BP present in these samples. Antioxidants such as vitamin E and BHA inhibited both lipid peroxidation and BP oxidation resulting from gamma-irradiation. These results demonstrate that the species generated during the peroxidation of unsaturated fats in foodstuffs can react with polycyclic aromatic hydrocarbons such as BP and convert them into active mutagenic and toxic products. This has important toxicological implications, particularly as the consumption of polyunsaturated fat in the Western world is increasing and gamma-irradiation may soon be widely used for food sterilization.