Singlet oxygen generation by UVA light exposure of endogenous photosensitizers

UVA light (320-400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), beta-nicotinamide adenine dinucleotide (NAD), and beta-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (PhiDelta = 0.54 +/- 0.07), FMN (PhiDelta = 0.51 +/- 0.07), and FAD (PhiDelta = 0.07 +/- 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue).     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Superoxide converts indigo carmine to isatin sulfonic acid: implications for the hypothesis that neutrophils produce ozone

Recently, it was proposed that neutrophils generate ozone (Wentworth, P. J., McDunn, J. E., Wentworth, A. D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J. M., Gutierrez, A., Janda, K. D., Babior, B. M., Eschenmoser, A., and Lerner, R. A. (2002) Science 298, 2195-2199; Babior, B. M., Takeuchi, C., Ruedi, J., Gutierrez, A., and Wentworth, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 3031-3034). Evidence for the proposal was based largely on the chemistry of ozone reacting with indigo carmine to produce isatin sulfonic acid. In this investigation, we have examined the specificity of this reaction and whether it can be used as unequivocal evidence of ozone production by neutrophils. Stimulated neutrophils promoted the loss of indigo carmine and formation of isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase. Methionine, which scavenges ozone, singlet oxygen, and hypochlorous acid, had no effect on the reaction. Neither did catalase or azide, which scavenge hydrogen peroxide and inhibit myeloperoxidase, respectively. From these results, it is apparent that superoxide was responsible for bleaching indigo carmine. Superoxide generated using xanthine oxidase and acetaldehyde also converted indigo carmine to isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase and unaffected by catalase. When the xanthine oxidase reaction was carried out in H(2)(18)O, the proportion of (18)O incorporated into the isatin sulfonic acid was the same as that found for ozone. Thus, reactions of ozone and superoxide with indigo carmine are indistinguishable with respect to isatin sulfonic acid formation. We conclude that bleaching of indigo carmine cannot be used to invoke ozone production by neutrophils. Studies using indigo carmine to implicate ozone in other biological processes should also be interpreted with caution.     

Oxygen, oxysterols, ouabain, and ozone: a cautionary tale

The recent account of the oxidation of tissue cholesterol by ozone created in human arterial plaques by the oxidation of water by electronically excited (singlet) dioxygen depends on the identification of the oxysterols formed and on the presumption that they are formed uniquely by ozone action. The chief oxysterols found, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 3beta,5-dihydroxy-5beta-B-norcholestane-6beta-carboxaldehyde, were identified as their 2,4-dinitrophenylhydrazones by chromatographic properties and a single mass spectral ion m/z 597 interpreted as [M-H](-). Conventional identification procedures for oxysterols were not conducted. Accordingly, absent other evidence, error may exist; such errors are known in the literature. Moreover, the assertion that ozone be the only oxidant that could form the 5,6-secosterol aldehyde from cholesterol is unproven. Other equally novel unproven processes can be posed. The account of biological ozone mimics prior 30-year-old reports of singlet oxygen itself in biological systems. Lest a similar history develop for biological ozone three topics of steroid oxidation are here reviewed to aid in understanding the current matter. Caution in evaluating the account of biological ozone is warranted.     

Singlet oxygen production by soybean lipoxygenase isozymes

The oxidation of linoleic acid catalyzed by soybean lipoxygenase isozymes was accompanied by 1268 nm chemiluminescence characteristic of singlet oxygen. The recombination of peroxy radicals as first proposed by Russell (Russell, G.A. (1957) J. Am. Chem. Soc. 79, 3871-3877) is a plausible mechanism for the observed singlet oxygen production. Lipoxygenase-3 was the most active isozyme. Under the optimal aerobic conditions of p2H 7, 100 micrograms/ml lipoxygenase-3, 100 microM linoleic acid, 100 microM 13-hydroperoxylinoleic acid, and air-saturated buffer, the yield of singlet oxygen was 12 +/- 0.4 microM or 12% of the amount predicted by the Russell mechanism. High yields of singlet oxygen required the presence of 13-hydroperoxylinoleic acid. Systems containing lipoxygenase-2 and lipoxygenase-3 produced comparable yields of singlet oxygen without added 13-hydroperoxylinoleic acid, since the lipoxygenase-2 served as an in situ source of hydroperoxide. Lipoxygenase-1 was active only at low oxygen concentrations. Its singlet oxygen-producing capacity was greatly increased by the addition of acetone to the system. Lipoxygenase-2 did not produce detectable quantities of singlet oxygen.     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO)

The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems.     

DNA strand scission in E.coli by electronically excited state molecules generated by enzymatic systems

$\text{E.}\text{coli}$ containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D₂ system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in $\text{E.}\text{coli}$ cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O₂ system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis.     

Singlet oxygen production by human eosinophils

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.     

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs

The antioxidant nordihydroguaiaretic acid (NDGA) has recently become well known as a putative anticancer drug. In this paper, it was evaluated the in vitro peroxynitrite (ONOO(-)), singlet oxygen ((1)O(2)), hydroxyl radical (OH(v)), hydrogen peroxide (H(2)O(2)), superoxide anion and hypochlorous acid (HOCl) scavenging capacity of NDGA. It was found that NDGA scavenges: (a) ONOO(-) (IC(50) = 4 +/- 0.94 microM) as efficiently as uric acid; (b) (1)O(2) (IC(50) = 151 +/- 20 microM) more efficiently than dimethyl thiourea, lipoic acid, N-acetyl-cysteine and glutathione; (c) OH(v) (IC(50) = 0.15 +/- 0.02 microM) more efficiently than dimethyl thiourea, uric acid, trolox, dimethyl sulfoxide and mannitol, (d) (IC(50) = 15 +/- 1 microM) more efficiently than N-acetyl-cysteine, glutathione, tempol and deferoxamine and (e) HOCl (IC(50) = 622 +/- 42 microM) as efficiently as lipoic acid and N-acetyl-cysteine. NDGA was unable to scavenge H(2)O(2). In an in vivo study in rats, NDGA was able to prevent ozone-induced tyrosine nitration in lungs. It is concluded that NDGA is a potent in vitro scavenger of ONOO(-), (1)O(2), OH(v), and HOCl and is able to prevent lung tyrosine nitration in vivo.     

Effects of ozone on cyclooxygenase metabolites in the baboon tracheobronchial tree

Short-term exposure to 0.5 parts per million (ppm) ozone has been shown to cause an increase in respiratory resistance in primates that can be diminished by 50% with pretreatment with cromolyn sodium. Because of the known membrane-stabilizing effects of cromolyn and the resultant inhibition of mediator production, we hypothesized a role for the products of arachidonic acid (AA) metabolism in these events. We exposed five adult male baboons to 0.5 ppm ozone on two occasions, once with cromolyn pretreatment and once without. Pulmonary resistance (RL) was monitored and bronchoalveolar lavage (BAL) was performed before and after each exposure. The BAL was analyzed for a stable hydrolysis product of prostacyclin, 6-keto-prostaglandin (PG) F1 alpha, PGE2, a stable hydrolysis product of thromboxane (Tx) A2, TxB2, and PGF2 alpha. RL increased after ozone exposure (1.62 +/- 0.23 to 3.77 +/- 0.51 cmH2O.l-1.s, difference 2.15; P less than 0.02), and this effect was partially blocked by cromolyn (1.93 +/- 0.09 to 3.18 +/- 0.40 cmH2O.l-1.s, difference 1.25; P less than 0.02). The base-line levels of the metabolites of AA in the BAL were as follows (in pg/ml): 6-keto-PGF1 alpha 72.78 +/- 12.6, PGE2 145.92 +/- 30.52, TxB2 52.52 +/- 9.56, and PGF2 alpha 22.28 +/- 5.42. Ozone exposure had no effect on the level of any of these prostanoids (P = NS). These studies quantify the magnitude of cyclooxygenase products of AA metabolism in BAL from baboon lungs and demonstrate that changes in the levels of these mediators in BAL are not prerequisites for ozone-induced increases in respiratory resistance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

Singlet oxygen production by biological systems

Singlet oxygen (1 delta g) is a highly reactive, short-lived intermediate which readily oxidizes a variety of biological molecules. The biochemical production of singlet oxygen has been proposed to contribute to the destructive effects seen in a number of biological processes. Several model biochemical systems have been shown to produce singlet oxygen. These systems include the peroxidase-catalyzed oxidations of halide ions, the peroxidase-catalyzed oxidations of indole-3-acetic acid, the lipoxygenase-catalyzed oxidation of unsaturated long chain fatty acids and the bleomycin-catalyzed decomposition of hydroperoxides. Results from these model systems should not be uncritically extrapolated to living systems. Recently, however, an intact cell, the human eosinophil, was shown to generate detectable amounts of singlet oxygen. This result suggests that singlet oxygen may be shown to be a significant biochemical intermediate in a few biological processes.     

A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimer's disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC(50) of 8.9+/-1.29 microM (n=6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 microM CSeco for 24h, for instance, resulted in 30.8+/-3.28% apoptotic and 1.8+/-1.11% of necrotic cells as against DMSO controls that only showed 1.3+/-0.33% of apoptosis and 1.6+/-0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.     

NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.     

Photo- and thermal-oxidation studies on methyl and phenyl linoleate: anti-oxidant behaviour and rates of reaction

Photo-peroxidation of methyl and phenyl linoleate in methanol solutions at 25 degrees C, in the presence of methylene blue or 5,10,15,20-tetra(4-pyridyl)-porphyrin (TPP) as sensitisers of singlet oxygen, was found to proceed at more than 30 times the rate of the same polyunsaturated fatty acid (PUFA) ester species undergoing thermal-peroxidation in the bulk phase at 50 degrees C. The addition of anti-oxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) quench the thermal-oxidation effectively but appear to only partially inhibit the photosensitized peroxidation reactions. The kinetics of the overall peroxidation reactions were followed by ultraviolet spectroscopy, measurements of hydroperoxide concentration and by high performance liquid chromatography (HPLC). The photo-peroxidation reaction proceeds more rapidly in chloroform solution as the lifetime of singlet oxygen is shown to be over ten times longer in chloroform than methanol. The initial fast reaction kinetics of the photo-peroxidation reactions were evaluated using a pulsed laser technique to show that singlet oxygen reacts competitively with both the anti-oxidants and the polyunsaturated fatty acid ester. Second order kinetic rate constants (in the range 10(5)-10(7) dm(3) mol(-1) s(-1)) were evaluated for the reactivity of singlet oxygen with a range of anti-oxidants and a singlet oxygen quencher, and the results used to explain the effect of anti-oxidants at different concentrations on the rate of the linoleate photo-peroxidation reaction.