N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities.

Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 microM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 microM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Activity of soybean lipoxygenase in the absence of lipid hydroperoxide

Soybean lipoxygenase was assayed under conditions such that the concentration of the enzyme was in excess of the concentration of the substrate, arachidonic acid. Under these conditions, the concentration of lipid hydroperoxides present as contaminants in the substrate was negligible relative to the enzyme concentration, and the concentration of lipid hydroperoxide product could be determined accurately. The ferric form of the enzyme was observed to be fully active and to catalyze the oxidation of arachidonic acid at a near-diffusion-controlled rate, 1.4 X 10(7) M-1 s-1 at 0 degree C, at concentrations of lipid hydroperoxides as low as 5% of the enzyme concentration. From this, it can be concluded that the higher oxidation states that would be accessible by oxidation of Fe(III) by hydroperoxide are not required for catalysis by soybean lipoxygenase. Surprisingly, the activation of the ferrous form of the enzyme was also observed at insignificantly low lipid hydroperoxide concentrations. This activation presumably involves oxidation of the ferrous to the ferric form of the enzyme and must be more facile than has hitherto been reported. This result may rationalize previous reports that the ferrous and the ferric forms of the enzyme are both active.     

Mung bean lipoxygenase in the production of a C6-aldehyde. Natural green-note flavor generation via biotransformation

Mung bean was investigated as a novel source of lipoxygenase in the natural production of the green-note aroma compound hexanal. Lipoxygenase extracted from mung bean catalyzed the oxidative reaction of linoleic acid, after which the intermediate hydroperoxide compound was split via green bell pepper hydroperoxide lyase to produce hexanal. In comparison to soybean lipoxygenase, mung bean lipoxygenase was found to be a good substitute as it produced 15.4 mM (76% yield) hexanal while soybean gave 60% yield. The mung bean pH profile comprised a wide peak (optimum pH 6.5) representing lipoxygenase-2 and lipoxygenase-3 isozymes, whereas two narrower peaks representing lipoxygenase-1 and lipoxygenase-2/3 isozymes were observed for soybean (optimum pH 10). Extraction at pH 4.5 was preferred, at which specific lipoxygenase activity was also the highest.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

EPR investigation of the active site of recombinant human 5-lipoxygenase: inhibition by selenide

Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO. After oxidation by lipid hydroperoxides, 5LO exhibited axial EPR spectra typified by a signal at g = 6.2. However, removal of the lipid hydroperoxides, their metabolites, and the solvent ethanol from the samples resulted in a shift to more rhombic EPR spectra (g = 5.17 and g = 9.0). Thus, many features of 5LO and soybean lipoxygenase-1 EPR spectra were similar, indicating similar flexible iron ligand arrangements in these lipoxygenases. Selenide (1.5 microM) showed a strong inhibitory effect on the enzyme activity of 5LO. In EPR, selenide abolished the signal at g = 6.2, typical for enzymatically active 5LO. Lipid hydroperoxide added to selenide-treated 5LO could not reinstate the signal at g = 6.2, indicating an irreversible change of the coordination of the active site iron.     

Electrospray ionization mass spectrometry of soybean lipoxygenases: N-terminal acetylation, chemical modification, and solution conformation

Electrospray ionization mass spectrometry was used to examine both the covalent structure and solution conformation of the soybean lipoxygenases. The post-translational modifications of two lipoxgyenases were identified as N-terminal acetylations by tandem mass spectrometry of peptides generated by trypsin digestion. The N-terminal sequence suggests that the proteins were substrates for the plant homolog of the N-terminal acetyltransferase complex C in yeast. Analysis of samples of native lipoxygenase-3 produced ions corresponding within experimental error to the mass of the N-acetylated polypeptide and one iron atom. The precision of the measurements was within roughly 100 ppm for the 96,856 Da protein. This made it possible to detect the addition of a single oxygen atom to the enzyme in a chemical modification reaction with cumene hydroperoxide. The acid-induced denaturation of lipoxygenase-3, which was accompanied by nearly complete loss of catalytic activity, was observed below pH 3.5 with the appearance of ions in the mass spectrum derived from the apoprotein. There was no evidence for the loss of iron in the absence of unfolding. Solutions of lipoxygenase-3 incubated in 0.1M acetic acid produced ions with a novel charge state distribution suggesting a unique conformation. Circular dichroism measurements showed that the secondary structure features of the native protein were retained in the new conformation. Dynamic light scattering revealed that the new conformation was not a consequence of protein aggregation as the hydrodynamic radius of lipoxygenase-3 was significantly smaller in acetic acid solution than at pH 7.0. Remarkably, the enzyme incubated in acetic acid retained full catalytic activity.     

Effect of lipid hydroperoxide on lipoxygenase kinetics.

In order to investigate the activation of lipoxygenase and to clarify the role of the oxygenation product hydroperoxide in this process, the effect of 13-hydroperoxylinoleic acid (P, 0-35 microM) on linoleic acid (S, 1-80 microM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 microM O2 with rapid kinetic techniques. The following observations were made: (1) Iron(II) and iron(III) lipoxygenases are kinetically different: reactions started with the Fe(II) enzyme form show a lag phase, whereas iron(III) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [S] greater than 50 microM, the initial burst in the iron(III) lipoxygenase curves is still followed by a lag. The lag phase disappears completely only in the presence of micromolar quantities of P. (3) The approximate dissociation constants for S and P are 15 and 24 microM, respectively, 1 order of magnitude smaller than the corresponding values in the absence of oxygen. The observed kinetics are predicted by numerical integration of the rate equations of a model based on the single lipid binding site mechanism for the anaerobic lipoxygenase reaction [Ludwig et al. (1987) Eur. J. Biochem. 168, 325-337; Verhagen et al. (1978) Biochim. Biophys. Acta 529, 369-379]. A quasi-steady-state approximation of the model suggests that a high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.     

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.     

X-ray absorption spectroscopy of soybean lipoxygenase-1. Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site.

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 2-3 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X-ray absorption fine structure shows clear differences in active-site structure as a result of this conversion. Curve-fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active-site structure proposed earlier (6 +/- 1 N/O ligands at 0.205-0.209 nm with a maximum variance of 0.009 nm, including 4 +/- 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al-Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 70-76], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 +/- 1 N/O ligands at 0.200-0.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data.     

Products of linoleic hydroperoxide-decomposing enzyme of alfalfa seed

Alfalfa seeds and seedlings contain an enzyme that catalyzes a reaction with the 13- and 9-hydroperoxides of linoleic acid to form 13-hydroxy-10-oxo-trans-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid, respectively. When commercial lipoxygenase is used to generate the hydroperoxides, the above acids appear in about 2:1 proportions, respectively. The products of the action of the enzyme on the hydroperoxides were stabilized for analysis by reduction with H(2) and LiAIH(4). Trimethylsilyl derivatives of reduced products were analyzed by combined gas-liquid chromatography-mass spectrometry. Specific deuterium labeling permitted definite location of the oxo functions. (18)O(2) labeling experiments showed that the oxygens of both the oxo and the hydroxyl functions were derived from the hydroperoxide. Retention of both oxygens suggests that the reaction proceeds through a cyclic epiperoxide followed by a ketohydroxy-forming rearrangement. No products of hydroperoxide isomerase were found in reactions catalyzed by the crude enzyme from alfalfa seeds.     

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.