Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.     

Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat.

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.     

Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury

A single dose of CCl4 when administered to a rat produces centrilobular necrosis and fatty degeneration of the liver. These hepatotoxic effects of CCl4 are dependent upon its metabolic activation in the liver endoplasmic reticulum to reactive intermediates, including the trichloromethyl free radical. Positive identification of the formation of this free radical in vivo, in isolated liver cells and in microsomal suspensions in vitro has been achieved by e.s.r. spin-trapping techniques. The trichloromethyl radical has been found to be relatively unreactive in comparison with the secondarily derived peroxy radical CCl3O2., although each free radical species contributes significantly to the biological disturbances that occur. Major early perturbations produced to liver endoplasmic reticulum by exposure in vivo or in vitro to CCl4 include covalent binding and lipid peroxidation; studies of these processes occurring during CCl4 intoxication have uncovered a number of concepts of general relevance to free-radical mediated tissue injury. Lipid peroxidation produces a variety of substances that have high biological activities, including effects on cell division; many liver tumours have a much reduced rate of lipid peroxidation compared with normal liver. A discussion of this rather general feature of liver tumours is given in relation to the liver cell division that follows partial hepatectomy.     

Chromium (III) decreases carbon tetrachloride-originated trichloromethyl radical in mice.

Effects of the administration of trivalent chromium (Cr(III)) to mice and the activation of carbon tetrachloride (CCl4) to form trichloromethyl radicals (.CCl3) in the liver were studied. The lipid peroxidation in liver microsomes induced in vitro by CCl4 in the presence of NADPH was decreased by the preadministration of Cr(III) to mice. The activity of NADPH-cytochrome C reductase, which presumably catalyzes the formation of .CCl3 from CCl4 in liver microsomes, was depressed by Cr(III) administration and kept at a level lower than that of the control group for at least 2 hr after CCl4 dosing. Furthermore, the frequency of appearances of ESR signals of .CCl3 in the liver homogenate of mice 1 min after CCl4 administration was markedly decreased by Cr(III) preadministration, similarly to DL-alpha-tocopherol. These results suggest that Cr(III) preadministered to mice decreases the formation of .CCl3 from CCl4, an activating process of CCl4, in the liver, presumably by scavenging the radical.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Spin trapping evidence for alcohol-associated oxidative stress

The spin trapping method combined with EPR spectroscopy has been employed by a number of laboratories to study alcohol-induced oxidative stress. Ethanol is converted to a free radical metabolite, the 1-hydroxyethyl radical in chemical reactions that generate hydroxyl radicals, in incubations of rat liver microsomes and in vivo. Furthermore, both acute and chronic ethanol administration leads to formation of radicals in the liver that are presumed to be products of lipid peroxidation. In general, overall radical production is enhanced by treatments known to be involved in alcoholic liver injury, such as chronic alcohol administration along with high levels of dietary fat.     

C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro

C-Phycocyanin (from Spirulina platensis) effectively inhibited CCl(4)-induced lipid peroxidation in rat liver in vivo. Both native and reduced phycocyanin significantly inhibited peroxyl radical-induced lipid peroxidation in rat liver microsomes and the inhibition was concentration dependent with an IC(50) of 11.35 and 12.7 microM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. These studies have demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC(50) of 5.0 microM and the rate constant ratios obtained for phycocyanin and uric acid (a known peroxyl radical scavenger) were 1.54 and 3.5, respectively. These studies clearly suggest that the covalently linked chromophore, phycocyanobilin, is involved in the antioxidant and radical scavenging activity of phycocyanin.     

Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane.

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.     

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.     

Inhibition of radical adduct reduction and reoxidation of the corresponding hydroxylamines in in vivo spin trapping of carbon tetrachloride-derived radicals.

In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.     

Detection of free radical formation in various tissues after acute carbon tetrachloride administration in gerbil

alpha-phenyl N-tert-butyl nitrone (PBN) was used as a spin-trapping agent to search for free radical formation in various tissues of gerbils. A significantly large amount of free radicals was detected in liver, kidney, heart, lung and testis after a single intraperitoneal dose of CCl4. We have also detected free radical formation in the brain and blood, although the number of spins was considerably smaller than those found in the other tissues. Free radicals formed in brain tissue after an oxidative insult may give strong supporting evidence for the free radical theory of aging.     

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride.

ATP-dependent calcium uptake of rat liver microsomes is examined following ingestion of CC14 (2.5 ml/kg). Within 30 min there is an abrupt drop in calcium uptake activity of the liver microsomes. This activity remains down for 48 hours before slowly returning to normal levels. The effect is specific for CC14 as contrasted with CHC13 and CH2Cl2. The CCl4 does not affect similar calcium uptake activity of kidney microsomes. Calcium uptake activity of the liver mitochondria is unaffected. The first 12 hours after CCl4 ingestion there is a relatively slow rise in the calcium content of the liver tissue and mitochondria. After 12 hours a much larger influx of calcium into the tissue and the mitochondria takes place. Forty-eight hours after CCl4 ingestion the process begins to slowly reverse. The following postulated sequence may relate to the CCl4 hepatotocicity. CCl4 is activated to free radicals by the liver endoplasmic reticulum. The free radical inactivate calcium pump activity of the liver endoplasmic reticulum. Calcium levels of the cytoplasm increase and significantly modify ion permeability of the plasma membrane. High levels of external calcium enter the cytoplasm and are sequestered in the mitochondria. The high level of mitochondrial calcium uptake inhibits mitochondrial oxidative phosphorylation. The specific sensitivity of the calcium pump activity of liver microsomes to CCl4 further establishes the identity of a system seperate from the mitochondrial system. The above postulated sequence of events would suggest a critical role in liver metabolism for calcium pump activity of the endoplasmic reticulum.     

Antioxidative effect of o-benzoquinone derivatives during CCl4-induced lipid peroxidation in the rat liver

It was found that o-benzoquinones (oBQ) inhibit the CCl4-dependent lipid peroxidation (LPO) in rat liver microsomes in vitro. The experimental data suggest that the antioxidant effect of oBQ is not due to the ability of these substances to shunt the NADPH-dependent electron transport pathways. More likely, oBQ inhibit LPO due to the ability of their reduced forms to scavenge the free radicals which induce LPO. Based on the experimental data, it was concluded that the increasing absorption of liver lipids at 230-236 nm after administration of CCl4 is due to the accumulation of reduced hydroperoxides. This process was shown to be inhibited by oBQ.     

Hepatoprotective activity of ellagic acid against carbon tetrachloride induced hepatotoxicity in rats

Administration of CCl4 to normal rats and consequent oral feeding with ellagic acid (50 mg/kg) provided a significant protection against the biochemical alterations in serum and liver produced by CCl4. In vitro experiments showed that liver microsomes from animals treated with ellagic acid and CCl4, decreased lipid peroxidation compared to microsome prepared from rats exposed to CCl4 alone.