Aldo Tomasi

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.

Hydroxyl free radical production in iron-cysteine solutions and protection by zinc

Abstract Hydroxyl radicals (OH − ) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.

Spin trapping of free radical products of CCI4 activation using pulse radiolysis and high energy radiation procedures

Free radicals have been suggested as activated intermediates in many types of tissue injury [ 1,2]. The techniques of electron spin spectroscopy (ESR) and pulse radiolysis are being increasingly used to characterise the free radicals involved, and to obtain quantitative data on their chemical reactivity [3,4]. Carbon tetrachloride is an important and much studied hepatoxin, which is metabolised by the NADPHcytochrome P450 electron-transport chain to an activated intermediate generally considered to be the trichloromethyl radical [5,6]. Attempts [7-91 to detect the Ccl; free radical in whole liver or liver fractions using direct ESR analysis have not been successful, and attention has consequently been directed to the less direct ESR spin trapping techniques [lo-121. Although this procedure is promising for studies both in vitro and in vivo, there are complications in interpretation of the ESR spectra of the free radical species produced in complex biomembrane environments [ 12,131. For this reason, we have studied the interaction of Ccl; with the spin-trap phenylbutylnitrone in a simplified model system where the Ccl; radicals are produced by a pulse of high energy electrons. We have previously obtained kinetic data on the reactivity of Ccl; and its peroxy-derivative CClaO; [14] in this model system. These data, together with ESR analysis with 13CC14 (as suggested in [l]), allow us to unequivocally identify the spintrap adducts of Ccl; and CCl,O;; identical spectra are also reported for microsomal systems, intact hepatocytes and under conditions in vivo.

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

The metabolism of halothane by hepatocytes: A comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.

Metabolic activation of 1,2‐dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes

A one‐electron reductive metabolism of 1,2‐dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using 13C‐DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.

Radiolysis of tetrachloromethane

Electron spin resonance studies of tetrachloromethane after exposure to 60Co γ-rays at 77 K reveal the formation of ·CCl3 and CCl˙+4 radicals. On warming in the presence of spin-traps, or on irradiating fluid solutions, nitroxide radical adducts have been detected that are characteristic of ·CCl3 and chlorine atom adducts. In the light of this evidence and that of other investigators a mechanism for the radiolysis of tetrachloromethane is postulated.In the presence of oxygen, ·CCl3 radicals are converted into Cl3COO· radicals. The use of spin-traps to detect these radicals is described and evaluated.

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes.

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.

ESR Spin-Trapping Artifacts in Biological Model Systems

The direct observation of free radicals in complex biological systems is often hampered because of the low concentration and high reactivity of the primary free radical species.* Spin trapping is a powerful tool that facilitates the visualization of free radicals, including those formed in such complex biosystems. The spin trap is a diamagnetic compound that reacts with a reactive free radical to form a more stable radical adduct. Although detection through electron-spin resonance spectroscopy offers several distinct advantages in its high sensitivity, and in some cases its specificity toward various radical species, there are also several drawbacks to using this technique.

Lipid Peroxidation and Bioactivation of Halogenated Hydrocarbons in Rat Liver Mitochondria During Experimental Siderosis

It is firmly extablished that increased amount of iron accumulated in hepatic parenchymal cells is associated with tissue injury, fibrosis and ultimately, cirrhosis1. However, the pathogenetic mechanism of iron in determining the liver injury has not been experimentally proven2. Currently two hypothesis have been put forward in order to explain the hepatocellular injury in chronic iron overload. The first implies that the excess iron largely occurring in lysosomes, physically disrupts these organelles with the release of cell damaging hydrolytic enzymes3. The second one presupposes that the pathological accumulation of iron elicits membrane lipid peroxidation in cellular organelles resulting in structural and functional alterations of cell integrity4. Indeed, iron could promote the production of reactive oxygen species such as Superoxide anion (O·− 2), hydroxy radicals (OH·− 4), and hydrogen peroxide (H2O2)5. The experimental evidence, gathered up to now, indicates that chronic iron overload may induce in vivo lipid peroxidation of mitochcndrial membranes6–8. Furthermore, the in vivo occurrence of lipid peroxidation in the mitochondrial membranes has been suggested to be responsible for some anomalies in liver mitochondria isolated from rats made siderotic either by dietary iron9,10 or by intraperitoneal injection of iron-(III)- gluconate complex11–13.