Girard's reagent P derivative of beta-Apo-8'-carotenal: a potent photoprotective agent.

A cationic carotenoid derivative (GRP-carotenal) was synthesized by the reaction of Girard's reagent P and beta-apo-8'-carotenal. The singlet-oxygen quenching constants for GRP-carotenal were 1.3 +/- 0.1 x 10(10) and 1.0 +/- 0.1 x 10(10) M-1 s-1 in acetonitrile and in detergent micelles, respectively. Photosensitized damage to K562 leukemia cells from cis-di(4-sulfonatophenyl)diphenylporphine, hypericin and protoporphyrin IX was inhibited by GRP-carotenal under conditions where beta-apo-8'-carotenal, beta-carotene and crocetin were ineffective. The unique cytoprotective properties of GRP-carotenal, relative to the other carotenoids studied, could not be explained by the differences in the cell content of the various carotenoids or by the changes in the cell content of the photosensitizers used. Photosensitizer fluorescence from labeled K562 cells was reduced by GRP-carotenal but not by the other carotenoids studied. The novel photoprotective properties of GRP-carotenal may be due to its subcellular distribution. In photosensitizer-containing detergent micelles, novel properties of GRP-carotenal were not apparent. None of the carotenoids studied reduced photosensitizer fluorescence or singlet-oxygen generation. Singlet-oxygen quenching by GRP-carotenal and by beta-apo-8'-carotenal were roughly the same. Crocetin has a singlet-oxygen quenching constant that is about a factor of five lower. Singlet-oxygen quenching by beta-carotene was limited by its aggregation.

Preferential cytotoxicity for multidrug-resistant K562 cells using the combination of a photosensitizer and a cyanine dye.

The cyanine dye 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (HIDC) protects K562 leukemia cells from photodynamic membrane damage caused by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and 420 nm light. This wavelength of light is chosen because it is absorbed by TPPS2, but not by HIDC. The photodynamic system studied may be useful as a model for antineoplastic therapy. A subline of K562 leukemia (K562/DOX), expressing the multidrug-resistance (MDR) phenotype, is found to accumulate smaller amounts of HIDC than the parent cell line and thus has less photoprotection. In the absence of added HIDC, the K562/DOX cell line is more resistant to photodynamic cytotoxicity than the K562 cell line. The resistance of the K562/DOX cell line is not due to a smaller accumulation of TPPS2 than the K562 cell line. However, when both cell lines are incubated with HIDC and TPPS2, and then exposed to light, the K562/DOX cell line becomes more sensitive to photodynamic cell damage than the K562 cell line. The combination of a photosensitizer with a cationic or lysomorphotropic photoprotector represents a novel strategy for the eradication of malignant cells expressing the MDR phenotype.

Cyanine dyes as protectors of K562 cells from photosensitized cell damage.

Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1',3,3,3',3'-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.

Inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Inactivation of glucose oxidase occurred in the presence of bromide, vanadate, H(2)O(2), and phosphate (the bromide system), and this was prevented by NADH or phenol red, a bromine acceptor. Glucose oxidase present during the reaction between diperoxovanadate and a reduced form of vanadate, vanadyl (the vanadyl system), but not added after mixing the reactants, was inactivated, and this was accompanied by a loss of binding of the dye, Coomassie blue, to the protein. The transient intermediate of the type OVOOV(O(2)), known to form in these reactions and used in the oxidation of bromide ion and NADH, appears to be responsible for inactivating glucose oxidase. In both systems, the inactivation of the enzyme was prevented by histidine and DTT, known to quench singlet-oxygen. By direct measurement of 1270-nm emission of singlet-oxygen, its generation was demonstrated in the bromide system, and in the reaction of hypohalous acids with diperoxovanadate, but not in the vanadyl system. By themselves both hypohalous acids, HOCl and HOBr inactivated glucose oxidase, and their prior reaction with H(2)O(2) during which singlet-oxygen was released, protected the enzyme. The results provide support for possible oxidative inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Singlet oxygen-mediated inactivation of acetylcholinesterase: a comparison of purified enzyme in solution and enzyme bound to K562 leukemia cells.

We have compared the singlet oxygen-mediated inactivation of acetylcholinesterase (ACE) in solution with the inactivation of ACE on the surface of K562 leukemia cells. In solution, the actions of the singlet-oxygen quenchers, methionine, azide, disodium [N,N'-ethylenebis (5-sulfosalicylideneimminato)]nickelate(II) (Ni-chelate 1) and disodium [(N,N'-2,3-propionic acid)bis(5-sulfosal-icylideneimminato)] nickelate(II) (Ni-chelate 2) could be explained quantitatively by assuming their only mechanism of action was to quench singlet oxygen. The singlet oxygen quenchers, azide, Ni-chelate 1 and Ni-chelate 2, caused smaller inhibitions in the rate of singlet oxygen-mediated inactivation of ACE on K562 cells than ACE in solution. The effects of these quenchers and of deuterium oxide were interpreted using a mathematical model of singlet-oxygen quenching and diffusion to estimate the lifetime of singlet oxygen near the cell surface. The azide quenching data and the deuterium-oxide data gave lifetimes of 0.9 +/- 0.2 microsecond and 0.45 +/- 0.15 microsecond, respectively. The increases in ACE inactivation lifetime caused by the nickel chelates were anomalously large. The unexpectedly large quenching due to the nickel chelates may have been due to a nonuniform distribution of the chelates in the cytoplasm with a large concentration of the chelate near the cell membrane.

On the synthesis of neurotransmitter receptor agonists with antagonist potential.

The synthesis of a new type of antagonist is described, capable of inactivating neuroreceptors with heretofore unattainable selectivity and permanence. These antagonists are referred to as mazek agonists (i.e. direct, inhibitory agonists) as they have the high receptor affinity and initial receptor-stimulatory effect of direct agonists and are positively coupled to effector systems. However, like direct antagonists, they have a high receptor affinity and the potential to inhibit or prevent receptor stimulation. The synthesis of the present compounds consisted of the covalent attachment of a tethered dye to three different neurotransmitter analogues, resulting in dye-neuropeptide conjugates with a high affinity for the FMRFa receptor. The dye was prepared from azure B (Az), the neurotransmitter was the neuropeptide FMRFamide (FMRFa), and the dye-neuropeptide conjugates synthesized were Az-CFMRFa; Az-CFMRF and Az-CLRFa. In this procedure, the analogues serve as carrier molecules, bound at one end to the receptor and at the other end to the dye, which is thereby brought into close contact with the receptor. The receptor can then be inactivated by singlet oxygen generated by laser irradiation of the photosensitized receptor.

Receptor inactivation by dye-neuropeptide conjugates. 3. Comparative binding of dye-neuropeptide conjugates to FMRFamide receptors of Helix aspersa and Loligo pealei.

Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [125I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [125I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC50 of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.

Receptor inactivation by dye-neuropeptide conjugates: 1. The synthesis of Cys-containing dye-neuropeptide conjugates.

In an attempt to attenuate specifically identified receptors through photolysis, a four-step synthesis is of a useful tethered derivative of Azure-B (Az) was developed After characterization, this derivative was covalently attached to CFMRFamide, CFMRF, and CLRFamide (i.e., three different neuropeptide analogues of the putative neurotransmitter FMRFamide. This resulted in the formation of three dye-neuropeptide conjugates: Az-CFMRFamide, Az-CFMRF, and Az-CLRFamide.

Receptor inactivation by dye-neuropeptide conjugates: 2. Characterization of the quantum yield of singlet oxygen generated by irradiation of dye-neuropeptide conjugates.

Different neuropeptide analogues of the neurotransmitter FMRFamide were covalently attached to a tethered dye, forming dye-neuropeptide conjugates capable of stably binding to the FMRFamide receptors. Singlet oxygen (1 delta O2) generated by laser irradiation of the conjugates bound to this receptor should inactivate it if (a) the distance 1 delta O2 must diffuse to reach the photo-sensitized receptor is less than 1000 A, (b) the conjugate binds the receptor with the same affinity as the indigenous neurotransmitter, and (c) the quantum yield (phi) of 1 delta O2 is sufficient. Previous studies determined that the first two constraints are satisfied. The results of the present study confirm that the third constraint is also satisfied, as the phi of 1 delta O2 resulting from the laser irradiation of the conjugates were uniformly large, exceeding those for the dye itself, ranging from 0.25 at pD 6.0 to 0.93 at pD 9.0.

Singlet oxygen generation from the reaction of ozone with plant leaves.

Aqueous extracts of the intercellular fluid from Sedum album L. leaves generated singlet oxygen chemiluminescence at 1270 nm when exposed to a nitrogen gas stream containing ozone at 21 +/- 2 ppm. The concentration of ascorbic acid in the intercellular fluid extracts was 310 +/- 40 microM. The intensity of the singlet oxygen chemiluminescence from the intercellular fluid extracts was comparable with the chemiluminescence from a control solution containing 300 microM ascorbic acid. The intensity of the singlet oxygen emission from intercellular fluid treated with ascorbate oxidase was 0.19 +/- 0.07 of the intensity of the singlet oxygen chemiluminescence from untreated samples of intercellular fluid extract. The simplest explanation for the effect of ascorbate oxidase is that ascorbic acid is the major ozone target generating singlet oxygen. Much weaker singlet oxygen chemiluminescence was detected at 1270 nm when intact S. album L. plant tips were exposed to a nitrogen gas stream containing ozone at 22 +/- 5 ppm. Various explanations for the relatively low intensity of the singlet oxygen chemiluminescence from intact S. album L. plant tips are discussed.

Reactive absorption of ozone by aqueous biomolecule solutions: implications for the role of sulfhydryl compounds as targets for ozone.

The rates of reactive absorption of ozone by various biomolecule solutions were measured. At pH 7, the ability of various biomolecules to reactively absorb ozone was in the following sequence: thiosulfate > ascorbate > cysteine approximately methionine > glutathione. The rates of reactive absorption under a variety of conditions were then analyzed using a mathematical model in order to estimate the reaction rate constants for the various ozone-biomolecule reactions. Compared to the ozone-methionine rate constant, the relative rate constants for thiosulfate, ascorbate, cysteine, and glutathione reactions were 18 +/- 2, 12 +/- 1, 1.1 +/- 0.1, and 0.62 +/- 0.03, respectively. Using an ozone-methionine reaction rate constant of 4 x 10(6) M-1 s-1, the rate constants for thiosulfate, ascorbate, cysteine, and glutathione were 7.2 x 10(7), 4.8 x 10(7), 4.4 x 10(6), and 2.5 x 10(6) M-1 s-1, respectively. Competitive studies using tryptophan as a standard ozone target were consistent with these rate constants. Compared to tryptophan, the relative ozone reaction rate constants for methionine, cysteine, and glutathione were 0.77 +/- 0.08, 0.88 +/- 0.19, and 0.42 +/- 0.01, respectively. These relative rate constants refer to ozone consumption rather than biomolecule consumption, so they may be compared with the relative rate constants obtained from reactive absorption. In addition, various inconsistencies in the literature regarding the rates of the ozone-cysteine and the ozone-glutathione reactions were reviewed.

Singlet oxygen chemiluminescence at gas-liquid interfaces: theoretical analysis with a one-dimensional model of singlet oxygen quenching and diffusion.

Singlet oxygen chemiluminescence was measured from the reaction of ozone with several biological molecules using a two-phase system in which ozone gas flowed over aqueous solutions of various biological molecules. Most of the chemiluminescence came from singlet oxygen which had diffused back into the gas phase. The intensity of the singlet oxygen chemiluminescence was adequately described by a one-dimensional mathematical model of singlet oxygen quenching and diffusion for the reaction of ozone with ascorbic acid, reduced glutathione, oxidized glutathione, methionine and NADH. Potentially, the theoretical model described in this paper could be used to interpret the singlet oxygen emission from complex biological systems with gas-liquid interfaces, such as ozone-exposed lungs or ozone-exposed plant leaves.

Singlet-oxygen generation at gas-liquid interfaces: a significant artifact in the measurement of singlet-oxygen yields from ozone-biomolecule reactions.

Several ozone-biomolecule reactions have previously been shown to generate singlet oxygen in high yields. For some of these ozone-biomolecule reactions, we now show that the apparent singlet-oxygen yields determined from measurements of 1270 nm chemiluminescence were artifactually elevated by production of gas-phase singlet oxygen. The gas-phase singlet oxygen results from the reaction of gas-phase ozone with biomolecules near the surface of the solution. Through the use of a flow system that excludes air from the reaction chamber, accurate singlet-oxygen yields can be obtained. The revised singlet-oxygen yields (mol 1O2 per mol O3) for the reactions of ozone with cysteine, reduced glutathione, NADH, NADPH, human albumin, methionine, uric acid and oxidized glutathione are 0.23 +/- 0.02, 0.26 +/- 0.2, 0.48 +/- 0.04, 0.41 +/- 0.01, 0.53 +/- 0.06, 1.11 +/- 0.04, 0.73 +/- 0.05 and 0.75 +/- 0.01, respectively. These revised singlet-oxygen yields are still substantial.

Time-resolved studies of singlet-oxygen emission from L1210 leukemia cells labeled with 5-(N-hexadecanoyl)amino eosin. A comparison with a one-dimensional model of singlet-oxygen diffusion and quenching.

Time-resolved measurements were made of near-infrared emission from 5-(N-hexadecanoyl)amino-eosin-labeled L1210 leukemia cells following pulsed-laser excitation. The cells were suspended in phosphate-buffered saline made with deuterium oxide solvent. A significant fraction of the emission occurring 10-80 microseconds after the laser pulse was due to singlet oxygen. This singlet-oxygen emission is believed to result from singlet oxygen generated near the cell-membrane surface, where 5-(N-hexadecanoyl)amino eosin is known to concentrate, and then diffusing out into the buffer. The intensity and the kinetics of the experimentally observed singlet-oxygen emission were in excellent agreement with the predictions of a theoretical one-dimensional model of singlet-oxygen diffusion and quenching. During the 10-80 microseconds time period studied, most of the singlet oxygen was located in the buffer. Thus, the use of water-soluble singlet-oxygen quenchers, such as histidine, provide one means of separating the singlet-oxygen emission from other sources of light during this time interval.

Mitomycin C-induced colitis in rats: a new animal model of acute colonic inflammation implicating reactive oxygen species.

The mechanism of the tissue damage induced by colonic inflammation in ulcerative colitis is not established. We therefore developed and characterized a simple new rat model of acute colonic inflammation induced by a single systemic injection of mitomycin C. After an intraperitoneal injection of mitomycin-C, colon histologic examination revealed transient (3 to 14 days) diffuse, colonic inflammation and injury that, like human ulcerative colitis, was limited to the mucosal layer. The rest of the gastrointestinal tract was spared. Gut permeability, as measured by urinary excretion of orally administered lactulose and mannitol, was unchanged 3 days after injection, when inflammation was already present; permeability was increased at 7 days, when inflammation was maximal. Mitomycin C did not produce inflammation in experimentally bypassed segments of small bowel despite the presence of colonic-type bacteria, suggesting that lack of intraluminal bacteria was not responsible for the absence of inflammation in the small intestine. Chemiluminescence, a means of estimating levels of reactive oxygen species, was greater in the intact, inflamed colon of mitomycin C-treated rats than in bypassed segments. Moreover, inflamed mucosal scrapings produced more in vitro luminol-enhanced chemiluminescence. Furthermore, the reactive oxygen species scavengers allopurinol, catalase, and WR-2721 decreased inflammation severity. We therefore conclude: (1) the mitomycin C-treated rat is a novel, easy to prepare animal model of acute inflammation of colonic mucosa, with morphologic similarities to the acute phase of ulcerative colitis in human beings; (2) increased gut permeability in mitomycin C-treated rats is the result, not the cause, of the inflammation; and (3) reactive oxygen species play an important role in colonic inflammation and tissue injury in this model, and possibly in human ulcerative colitis.

Quenching of singlet oxygen by biomolecules from L1210 leukemia cells.

Singlet oxygen lifetimes for detergent-dispersed L1210 leukemia cells in deuterium oxide buffer were measured by following the decay of 1270 nm phosphorescence. Four photosensitizers and two detergents were studied. Stern-Volmer plots were linear over the cell concentration range studied (0-10(7) cells/mL). The singlet-oxygen quenching constants obtained depended somewhat upon the specific combination of detergent and photosensitizer used. Extrapolation of the singlet-oxygen lifetime data to "100%" cell concentration (1.39 +/- 0.04 x 10(9) cells/mL) and correction for the contribution of the water solvent gave a singlet-oxygen lifetime between 0.17 and 0.32 microseconds for the L1210 leukemia cell. The theoretical contributions of various types of biological molecules within the L1210 cell to the total singlet-oxygen quenching were calculated from their concentrations and their quenching constants. These calculations suggest that proteins will quench most of the singlet-oxygen. Only about 7% of the singlet-oxygen is quenched by water.

Singlet oxygen production from the reactions of ozone with biological molecules.

The reaction of ozone with a number of biological molecules was found to produce singlet oxygen in high yield. At pH 7.0, the reaction of ozone with an equimolar amount of biological molecule produced the following singlet oxygen yields (mole of singlet oxygen/mole of ozone): cysteine, 0.49 +/- 0.02; methionine, 1.13 +/- 0.11; reduced glutathione, 0.33 +/- 0.02; albumin, 1.00 +/- 0.05; uric acid, 0.64 +/- 0.09; ascorbic acid, 0.96 +/- 0.007; NADPH, 1.07 +/- 0.07; NADH, 0.95 +/- 0.01. Thus, singlet oxygen may be an important intermediate in the biochemical damage caused by ozone.