Thiyl radicals in biosystems: effects on lipid structures and metabolisms.

Thiyl radicals are intermediates of enzyme- and radical-driven biochemical processes, and their potential as reactive species in the biological environment has been somehow underestimated. From organic chemistry, however, it is known that thiyl radicals isomerize the double bonds of unsaturated fatty acids to a mixture with very dominating trans isomers. Recently, this reaction has been particularly studied for biosystems, focusing on the effect of thiyl radicals on the natural all-cis double bonds of unsaturated phospholipids, which undergo a conversion to the unnatural trans form. In this paper we report briefly the role of thiyl radicals in biosystems, describe the main features of the radical-induced cis-trans isomerization process under both in vitro and in vivo conditions, and reflect on some consequences for membrane structures, lipid metabolism and enzymatic reactions.

Radiation-induced fragmentation of cardiolipin in a model membrane.

PURPOSE: To obtain evidence for the possibility of free-radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes. MATERIALS AND METHODS: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to gamma-rays from 60Co or 137Cs sources at doses between 1 and 24kGy. Fragmentation products were identified using thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Using MALDI-TOF MS and thin-layer chromatography, it was shown that gamma-irradiation of liposomes consisting of TMCL was accompanied by free-radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen. CONCLUSION: It is shown for the first time that on gamma-irradiation, cardiolipin can undergo free-radical fragmentation in its polar component.

Efficiency and mechanism of the antioxidant action of trans-resveratrol and its analogues in the radical liposome oxidation.

trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a nonflavonoid polyphenol reported to exert different biological activities, among them inhibition of the lipid peroxidation, scavenging of the free radicals, inhibition of the platelet aggregation, and anticancer activity as the most important. In order to enlighten the radical-scavenging mechanism of trans-resveratrol, stationary gamma-radiolytic experiments in liposomes and pulse radiolytic experiments in aqueous solutions were performed. Applying the stationary gamma-radiolysis together with the subsequent product analysis, reactions of lipid peroxyl radicals, LOO*, with trans-resveratrol and other natural antioxidants were investigated. It was found that trans-resveratrol was a better radical scavenger than vitamins E and C but similar to the flavonoids epicatechin and quercetin. The comparison of the radical-scavenging effects of trans-resveratrol and its analogues trans-4-hydroxystilbene and trans-3,5-dihydroxystilbene revealed that trans-resveratrol and trans-4-hydroxystilbene showed almost the same effect and were more efficient than trans-3,5-dihydroxystilbene. These findings indicate greater radical-scavenging activity of trans-resveratrols para-hydroxyl group than its meta-hydroxyl groups. Using the pulse radiolysis, reactions of trans-resveratrol and its analogues with trichloromethylperoxyl radicals, CCl(3)OO*, were studied. Spectral and kinetic properties of the observed transients showed great similarity between trans-resveratrol and trans-4-hydroxystilbene which seems to confirm that para-hydroxyl group of trans-resveratrol scavenges free radicals more effectively than its meta-hydroxyl groups.

Photoinduced chain reactions of alcohols in the presence of diphenyliodonium ion pairs with cyanometallates--steady state UV/visible spectroscopic and pulse radiolysis studies.

Diphenyliodonium ions (Ph2I+) form donor-acceptor ion pairs with suitable cyanometallates such as [Mo(CN)8]4-, [W(CN)8]4-, [Ru(CN)6]4- and [Os(CN)6]4-. Such ion pairs are characterized by new spectroscopic transitions due to second-sphere interactions between donor ([M(CN)x]4-, x=6, 8) and acceptor (Ph2I+) ions. Photochemical excitation of these ion-pair charge-transfer (IPCT) states leads to efficient electron transfer reactions that yield short-lived diphenyliodyl radicals (Ph2I*) and oxidized cyanometallates ([M(CN)x]3-). Diphenyliodyl radicals decay to iodobenzene and phenyl radicals. This very convenient source for generating phenyl radicals was applied to the photoinduced chain oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, respectively. However, unexpected side reactions led to undesired chain-terminating reactions. Adduct formation of diphenyliodonium ions with alpha-hydroxyalkyl radicals was verified by pulse radiolysis studies. These relatively longlived adducts give rise to chain-terminating reactions because of interactions with [M(CN)x]4- complexes that lead to oxidized cyanometallates [M(CN)x]3- upon regeneration of the starting alcohols.

Transformation of phospholipid membranes by thiyl radicals via cis-trans isomerization of fatty acid residues.

Thiyl radical-mediated transformation of unsaturated fatty acid residues are reported. Beside the initiation of lipid peroxidation, thiyl radicals can efficiently cause isomerization of fatty acid residues in a catalytic manner. The latter process is observed in homogeneous solutions as well as in organized assemblies, leading to a denaturation of the natural all-cis-isomers of fatty acid residues of lipid bilayers. The degree of denaturation goes parallel with an increase in the number of double bonds and in the hydrophobicity of thiols. For arachidonic acid methyl ester, a decay of the all-cis-isomer to approximately 30% after a gamma irradiation dose of 1.8 kGy in micelles is observed. The reversibility of the isomerization reaction is demonstrated in liposomes by the fact that the cis/trans-equilibrium is independent of configuration of the starting compound.

On the photochromism of spiro

The recently synthesized spiro[cyclohexadiene-dihydroacridines] consisting of perpendicularly arranged aroylcyclohexadiene and N-methyl-dihydroacridine moieties were found to have photochromic properties. The reversible photoisomerization from the spiro compound toward a colored merocyanine caused by C-C bond cleavage in the cyclohexadiene was studied by stationary and time-resolved measurements of their optical spectra. The course of the absorption under UV and visible irradiation, respectively, and HPLC analysis of the photoproducts result in the determination of excitation energy-dependent quantum yields for the merocyanine formation and, in reverse, the ring closure, as well as degradation. Whereas the thermal back reaction completely recovers the spiro compound (k approximately 6.8 x 10(-4) s-1, T = 22 degrees C), degradation of the merocyanine under irradiation at 480 nm has a probability of about 6%. Picosecond-resolved measurements of the fluorescence and the transient absorption show that photoisomerization occurs via the first excited singlet state within 100 ps depending on the activation barrier.

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes.

NMR spectroscopy and gas chromatography were used on methanolic solutions of fatty acid methyl esters and on small bilayer liposomes to study the radical-induced denaturation of the fatty acid residues from the natural cis-configuration into trans-isomers. To analyze the mechanism of the thiyl radical-catalyzed lipid isomerization, we compared the effects of thiols on oleic and linoleic fatty acid residues using pulse radiolysis, gamma-radiolysis and chemolysis (AAPH) to generate thiyl radicals. The isomerization step takes place within the adduct of the thiyl radical to an olefinic group of unsaturated fatty acids, but not within the pentadienyl radical. The stability of the adduct can be described by an equilibrium constant of (12+/-5) mol(-1) dm(3). The isomerization rate depends on the structure of the thiol. However, the resulting isomeric equilibrium (trans-fraction: 81%) does not depend on the structure of the thiyl radical or the organization of the lipids. Quantum chemical calculations were performed to estimate the barriers for rotation, the geometry and the enthalpy difference between cis- and trans-thiyl radical adducts.

Differences in the reactivity of phthalic hydrazide and luminol with hydroxyl radicals.

The reactivity of 5-amino-2,3-dihydro-phthalazine-1,4-dione (luminol) and phthalic hydrazide with hydroxyl radicals was studied. HO*-radicals were generated by the Fenton reaction as well as by water radiolysis. Both luminol and phthalic hydrazide react with hydroxyl radicals under intense chemiluminescence (CL) emission. However, exclusively the CL arising from phthalic hydrazide oxidation can be quenched by competition (e.g. by the addition of carbohydrates), whereas luminol CL is enhanced. The reactivities of both compounds with HO*-radicals were further studied by time-resolved spectroscopy (pulse radiolysis), competition methods, NMR spectroscopy and mass spectrometry. Whereas only slight differences were detectable by pulse radiolysis, the analysis of competition kinetics in the presence of p-nitroso-dimethylaniline (NDMA) gave a two-fold-enhanced reactivity for luminol (4.8 x 10(9) l mol(-1) s(-1)) in comparison to phthalic hydrazide (2.0 x 10(9) l mol(-1) s(-1)). NMR and mass spectrometric analyses revealed significant differences in the reactivity of HO*-radicals: whereas in luminol solutions hydroxylation of the aromatic ring system predominated, hydroxylated products were not detectable upon irradiation of phthalic hydrazide. A hypothetical mechanism is proposed which may explain the observed differences.

Thiyl radical-induced cis/trans-isomerization of methyl linoleate in methanol and of linoleic acid residues in liposomes.

PURPOSE: To investigate the role of a thiol-containing biologically active compound in lipid peroxidation of membranes. MATERIALS AND METHODS: Thiyl radicals were generated from 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)-dione (MECH) using pulse radiolysis and gamma-radiolysis in aqueous and alcoholic solutions saturated with N2O. The products were analysed by 1H NMR and by HPLC. RESULTS: THE thiyl radicals abstract bisallylic hydrogens from [cis-9, cis-12]-methyl linoleate, yielding a pentadienyl radical. In the absence of oxygen, a thiyl radical-induced cis/trans-isomerization leads to linoleic-type isomers. These chain-type isomerization reactions can occur with the long living pentadienyl radical, followed by a 'repair' reaction of the attached thiol, and with the thiyl radical adduct with a double bond of the fatty acid residue. CONCLUSIONS: The results show that the mechanism of cis/trans-isomerization is an integral part of the thiyl radical attack on polyunsaturated fatty acids in homogeneous solutions and in bilayers.

Reactivity of cartilage and selected carbohydrates with hydroxyl radicals: an NMR study to detect degradation products.

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by gamma-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosaccharides as well as carbon resonances in 13C-enriched glucose were continuously diminished upon gamma-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) gamma-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysaccharides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.

The reaction of pristane (2,6,10,14-tetramethylpentadecane) with radiolytically generated reactive oxygen intermediates results in a stable genotoxic compound as assessed by the SOS chromotest.

The most widely studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p. injections of the isoalkane pristane (2,6,10,14-tetramethylpentadecane). Employing a simple quantitative and well-established short-term bacterial genotoxicity assay, the SOS chromotest, as a model system, we have investigated whether pristane may potentially be involved in causing or modulating the genotoxic events thought to induce plasma cell tumorigenesis. We found that incorporation of pristane into the cell membranes enhance the SOS response in Escherichia coli PQ37 and PQ300 induced by gamma-radiation under hyperoxic conditions. Moreover, the oxidation of pristane by radiolytically generated reactive oxygen intermediates yielded a stable, genotoxic product active on E. coli PQ300, a SOS tester strain designed to detect oxidative genotoxins. We discuss these findings in relation to the tumor-promoting role of the chronic i.p. inflammation that accompanies plasmacytomagenesis and conclude that, under these specific conditions, pristane may possess a previously unrecognized genotoxic activity in its tumorigenic potential.