Presence Of Deuterium Oxide Research Articles

Rapid Antibiotic Susceptibility Testing by Deuterium Labeling of Bacterial Lipids in On-Target Microdroplet Cultures.

Antimicrobial resistance is a serious challenge facing human and veterinary health. Current methods of detecting resistance are limited in turn-around time or universal detection. In this work, a new antimicrobial susceptibility test is developed and validated, which utilizes deuterium labeling of membrane lipids to track the growth of bacterial cells. We hypothesize that deuterium uptake and subsequent labeling of lipids can be detected using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Additionally, bacteria growth is performed on the MALDI target, minimizing sample preparation materials and time. When two Escherichia coli strains are grown in the presence of deuterium oxide, labeling can be detected in as little as 30 min to 2 h. The labeling efficiency, or the ratio of labeled to unlabeled lipid peaks, provides information about the growth rate of bacteria. This growth ratio can differentiate between resistant and susceptible strains of bacteria as a resistant strain will maintain ∼50% labeling efficiency between untreated and treated cultures. In comparison, a susceptible strain will see a decrease in fractional abundance of deuterium from ∼50% in the untreated to ∼10% in the treated. This approach is applied to measure the minimum inhibitory concentration (MIC) of the resistant and susceptible strains from on-target microdroplet culture in a range of antibiotic concentrations. The first antibiotic concentration with a significant decrease in fractional abundance of deuterium correlates well with a traditionally obtained MIC using broth dilution, indicating the clinical relevance of the results.

Mechanistic Insight into the Catalytic Electroreduction of Tetrabromobisphenol-a at Carbon Cathodes

Polyhalogenated phenols are a class of environmental pollutants that serve as precursors to dioxins and behave as endocrine disruptors.1,2 As a result, an efficient route of degradation is needed. Traditional methods of remediation include microbial, thermal, and chemical pathways; however, these typically require large amounts of toxic solvents, excessive amounts of energy, and can be tremendously time-consuming.3 Electrochemistry has become a growing area of research for the remediation of environmentally-harmful compounds, as the electroreductive behavior of carbon–halogen bonds is well understood.Herein, we report the electrochemical reduction of the flame retardant 3,3',5,5'-tetrabromobisphenol-A (TBBPA), at carbon cathodes in the absence and presence of nickel(II) salen. Cyclic voltammograms at varying scan rates, coupled with voltammograms in the presence of a hydrogen-bonding stabilizer, suggest reduction proceeds by cleavage of a carbon–bromine bond, followed by reduction of a short-lived intermediate. Controlled-potential (bulk) electrolysis was performed to determine the extent of reduction that could be achieved; moreover, gas chromatography–mass spectrometry was utilized to separate and identify reduction products. Electrolyses performed in the presence of deuterium oxide and 1,1,1,3,3,3-hexafluoroisopropanol-OD reveal a carbanion intermediate present in the reduction mechanism. References M. Montaño, A. C. Gutleb, and A. J. Murk, Environ. Sci. Technol., 2013, 47, 6071–6081.C. D. Klaassen, Casarett & Doull’s Toxicology the Basic Science of Poisons, McGraw-Hill Education, 2013.E. T. Martin, C. M. McGuire, M. S. Mubarak and D. G. Peters, Chem. Rev., 2016, 116, 15198–15234.

Functionalization of Alkenyl C–H Bonds with D2O via Pd(0)/Carboxylic Acid Catalysis

We report herein a simple catalytic method for the extensive labeling of alkenyl C–H bonds through the combination of a palladium(0) complex and a carboxylic acid in the presence of deuterium oxide. The reaction can be applied to a variety of terminal alkenes and the best results are obtained with aryl-substituted examples. This method represents a convenient approach for the preparation of extensively labeled chemicals from the cheapest and safest source of deuterium.

Use of selected scavengers for the determination of NF-TiO2 reactive oxygen species during the degradation of microcystin-LR under visible light irradiation

Although UV-induced TiO2 photocatalysis involves the generation of several reactive oxygen species (ROS), the formation of hydroxyl radicals is generally associated with the degradation of persistent organic contaminants in water. In this study, a variety of radical scavengers were employed to discriminate the roles of different ROS during visible light-activated (VLA) photocatalysis using nitrogen and fluorine doped TiO2 (NF-TiO2) in the degradation of the hepatotoxin, microcystin-LR (MC-LR) in water. The addition of hydroxyl radical scavengers, methanol and tert-butyl alcohol to the reaction mixture resulted in negligible inhibition of NF-TiO2 photocatalytic degradation of MC-LR at pH 3.0 and only partial inhibition at pH 5.7 under visible light. While hydroxyl radicals (OH) generally play the primary role in UV-TiO2 photocatalysis, the minimal influence of MeOH and t-BuOH on the degradation process under these experimental conditions indicates that OH are not crucial in VLA NF-TiO2 photocatalysis. However, strong inhibition was observed in VLA NF-TiO2 photocatalytic degradation of MC-LR in the presence of superoxide dismutase, benzoquinone and catalase at pH 3.0 and 5.7 indicating that O2− and H2O2 play critical roles in the degradation process. Similar degradation rates were observed in the presence of deuterium oxide, which enhances singlet oxygen mediated processes further suggesting singlet oxygen is not a key species in the degradation of MC-LR. Formic acid and cupric nitrate were added to probe the roles of the valence band holes and conduction band electrons, respectively. Under UV–vis light irradiation, almost complete inhibition of MC-LR removal is observed with NF-TiO2 in the presence of OH scavengers at pH 5.7. These results demonstrate that the solution pH plays a major role in the formation and reactivity of ROS during VLA NF-TiO2 photocatalysis. The adsorption strength of scavengers and MC-LR onto NF-TiO2 as well as the speciation of ROS as a function of pH needs to be carefully considered since they also play a major role in the efficiency of the process. These results indicate that the reduction of molecular oxygen by photo-generated electrons rather than hydroxyl radicals produced by oxidative reactions of photo-generated holes is the key factor in the VLA NF-TiO2 photocatalytic degradation of MC-LR.

Rapid and efficient desilylation and deuteration of alkynylpyridines

We describe a rapid and efficient technique for the direct installation of deuterium atoms following the removal of trimethylsilyl groups from alkynylpyridines. Utilizing tetrabutylammonium fluoride in the presence of deuterium oxide, we observe up to 97% deuterium incorporation in as little as one minute of reaction time.

Reduction of α,β- and α,ω-Dibromoalkanes at Silver Cathodes

Electrochemical reduction of halogenated organic pollutants via electrolysis at silver cathodes is a remediation strategy that has been undertaken by many laboratories, including our own, due to the catalytic capability of silver to cleave carbon–halogen bonds reductively. However, due to the complexity of these polyhalogenated compounds, their reduction mechanisms are not well understood and have, in turn, inspired us to map the reductions of various alkyl halides at silver.Past investigations of the electrochemical behavior of monohalides at silver cathodes have demonstrated that (a) identity of the halide, (b) position of the halide, and (c) dryness of the solvent–electrolyte [dimethylformamide (DMF) containing 0.050 M tetramethylammonium perchlorate (TMAP)] have an effect on the outcome of the reduction.1 Rondinini et al.2 reported that a second group with specific affinity for the silver surface increases the effects observed in cyclic voltammetry due to the structure of various alkyl halides. On the basis of this dependence, Rondinini and co-workers suggest that, in the case of an α,ω-dihaloalkane, both halogens are coadsorbed at silver; thus, intramolecular coupling should be favored. Our present work begins to illuminate how the relative positions of the halogens affect the reduction mechanisms of α,β- and α,ω-dibromoalkanes. Cyclic Voltammetry. Comparison of cyclic voltammograms for the reduction of 1,2-dibromohexane (1) and 1,6-dibromohexane (2) in DMF–0.050 M TMAP at a scan rate of 0.1 V s–1 reveals a single cathodic peak for each compound (Figure 1). Interestingly, the cathodic peak observed for reduction of 1 is shifted 0.68 V less negative and the peak current is approximately one-half of that observed for reduction of 2. Second, the cathodic peak for 2 is observed close to the cathodic peak potentials observed for 1- and 2-bromohexane. For both 1 and 2, a plot of peak current versus concentration for solutions between 0.5 and 20 mM at a scan rate of 0.1 V s–1 show behavior anticipated for a typical irreversible reaction; however, closer inspection reveals that at lower concentrations the current function (ip/(C0 *ν1/2)) increases, which can be attributed to an escalation of the relative contribution from reduction of adsorbed reactant. Cathodic peak currents obtained for reduction of 0.5, 2.0, and 8.0 mM solutions of 1 and 2 are linear with respect to the square root of the scan rate for moderate scan rates (0.05 to 1 V s–1); at higher scan rates (>10 V s–1), the peak currents no longer increase. Controlled-Potential (Bulk) Electrolyses. Bulk reduction of 1 and 2 at silver electrodes in DMF–0.050 M TMAP reveals that product distribution and reaction mechanism are dependent on the relative positions of the two bromine atoms. Reductions of 1 exhibit a second spike in the current–time (i–t) curve; on the other hand, reduction of 2 results in a single exponential decay in the i–t curve. Furthermore, the sole product of reduction of 1 is 1-hexene, whereas reduction of 2 affords hexane as the major product. Coulometric n values for the reduction of 1 indicate a two electron pathway; however, higher n values arise from electrolysis of 2. Electrolyses performed in the presence of a 100-fold excess of deuterium oxide give no evidence for the presence of carbanions during the reduction of 1; in contrast, monodeuterated hexane was detected after reduction of 2 in the presence of deuterium oxide. No monobrominated species were detected when 1 was electrolyzed exhaustively in the absence or presence of an excess of a proton donor (hexafluoroisopropanol or water). Moreover, no monobrominated product was detected when electrolysis of 1 was interrupted. References Strawsine, L. M.; Mubarak, M. S.; Peters, D. G. J. Electrochem. Soc. 2013, 160, G3030–G3037.Rondinini, S.; Mussini, P. R.; Muttini, P.; Sello, G. Electrochim. Acta 2001, 46, 3245–3258.

Multiscale analysis of water uptake and erosion in biodegradable polyarylates

The role of hydration in degradation and erosion of materials, especially biomaterials used in scaffolds and implants, was investigated by studying the distribution of water at length scales from 0.1 nm to 0.1 mm using Raman spectroscopy, small-angle neutron scattering (SANS), Raman confocal imaging, and scanning electron microscopy (SEM). The measurements were demonstrated using l-tyrosine derived polyarylates. Bound- and free- water were characterized using their respective signatures in the Raman spectra. In the presence of deuterium oxide (D2O), H-D exchange occurred at the amide carbonyl but was not detected at the ester carbonyl. Water appeared to be present in the polymer even in regions where there was little evidence for NH to ND exchange. SANS showed that water is not uniformly dispersed in the polymer matrix. The distribution of water can be described as mass fractals in polymers with low water content (∼5 wt%), and surface fractals in polymers with larger water content (15–60 wt%). These fluctuations in the density of water distribution are presumed to be the precursors of the ∼20 μm water pockets seen by Raman confocal imaging, and also give rise to the 10–50 μm porous network seen in SEM. The surfaces of these polymers appeared to resist erosion while the core of the films continued to erode to form a porous structure. This could be due to differences in either the density of the polymer or the solvent environment in the bulk vs. the surface, or a combination of these two factors. There was no correlation between the rate of degradation and the amount of water uptake in these polymers, and this suggests that it is the bound water and not the total amount of water that contributes to hydrolytic degradation.

Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA).

Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking. Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking. For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms. RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.

Moisture Adsorption in Plasma-Damaged Porous Low-k Dielectrics

ABSTRACTPlasma etch/ash processes can induce changes in low-k film surface/bulk chemistries and topographies resulting in increased water absorption, surface roughness, and metal intrusion. After ashing, the altered surface character of the low-k can impact wetting, adhesion, and, consequently, the resistance of subsequently deposited barrier layers. In this work, we describe the use of deuterium oxide as means of measuring moisture penetration into low-k films. Film chemistries have been monitored using grazing angle attenuated total reflectance (GATR) and transmission Fourier transform infrared spectroscopy (FTIR). To study moisture absorption in porous spin-on and CVD low-k films, unashed and ashed films have been exposed to D2O liquid and vapor treatments under “dry” nitrogen. The extent of D2O uptake, removal and exchange reactions has been studied using transmission and GATR FTIR methods because the D2O and O-D adsorption peaks are distinct from water and O-H as well as other low-k adsorptions. This method can be used to study Si-OH species because deuterium can exchange with hydrogen within silanols under ambient conditions while methyl groups are much less likely to exchange. Three different low-k films, a porous spin-on MSQ (k=2.2), a porous CVD (k=2.3), and an organosilicate glass (OSG, k=2.85) have been used. In FTIR spectra, unashed low-k films show minimal D2O adsorption. In MSQ hydrogen-ashed films, the data suggest the presence of deuterium oxide and O-D peaks. Further, D2O adsorption appears to be considerably higher for ashed films as would be expected due to the hydrophobicity of these films. In the CVD films, there does not appear to be as marked a difference. This method permits the introduction of a chemical “marker” into low-k wet and ambient processes allowing one to distinguish among adsorptions from different aqueous sources.

UVA Photoirradiation of Methylated Benzo[a]pyrene and Benzo[e]pyrene leading to Induction of Lipid Peroxidation

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants and potentially pose a health risk to humans. Although the biological and toxicological activities, including metabolism, mutagenicity and carcinogenicity of PAHs have been thoroughly studied, their phototoxicity and photo-induced biological activities have not been well examined. In this research, we studied the photoirradiation of isomeric methylbenzo[a]pyrene (MBaP) and methylbenzo[e]pyrene (MBeP) by UVA light in the presence of a lipid, methyl linoleate, and evaluated the potential of these compounds to induce lipid peroxidation. The compounds chosen for study included BaP, 3-MBaP, 4-MBaP, 6-MBaP, 7-MBaP, 10-MBaP, BeP, 4-MBeP, and 9-MBeP. The results indicate that upon photoirradiation by UVA at 7 and 21 J/cm2, these compounds induced lipid peroxidation. The levels of the induced lipid peroxidation were similar among BaP and the isomeric MBaPs, and among the BeP and MBePs, with the BaP group higher than the BeP group. There was also a co-relation between the UV A light dose and the level of lipid peroxidation induced. Lipid peroxide formation was inhibited by NaN3 (singlet oxygen and free radical scavenger) and was enhanced by the presence of deuterium oxide (D2O) (extends singlet oxygen lifetime). These results suggest that photoirradiation of MBaPs and MBePs by UVA light generates reactive oxygen species (ROS), which induce lipid peroxidation.

Photoirradiation of representative polycyclic aromatic hydrocarbons and twelve isomeric methylbenz[a]anthracene with UVA light: formation of lipid peroxidation

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants, which require metabolic activation in order to exert biological activities, including mutagenicity and carcinogenicity. Photoactivation is another activation pathway that can lead to PAH genotoxicity. In this paper, we demonstrate that photoirradiation of a series of representative PAHs, with and without bearing a methyl substituent, with UVA light in the presence of methyl linoleate resulted in the formation of methyl linoleate hydroperoxides (a lipid peroxide). The lipid peroxide formation was inhibited by dithiothreitol (DTT) (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), and superoxide dismutase (SOD) (superoxide scavenger), but was enhanced by the presence of deuterium oxide (D2O) (extends singlet oxygen lifetime). These results suggest that photoirradiation of PAHs by UVA light generates reactive oxygen species (ROS), which induce lipid peroxidation.

A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6.

A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.

Microwave mediated hydrogen deuterium exchange: a rapid synthesis of 2H-substituted benzimidazole

Deuterium aromatic substituted compounds were prepared by microwave irradiation of the parent unlabeled compounds in the presence of deuterium oxide and deuterium chloride. The percentage of deuterium incorporation was investigated under various reaction conditions. A rapid synthesis of 2H-substituted benzimidazole under microwave irradiation is described.

Photodecomposition of Retinyl Palmitate in Ethanol by UVA LightFormation of Photodecomposition Products, Reactive Oxygen Species, and Lipid Peroxides

Photodecomposition of retinyl palmitate (RP), an ester and the storage form of vitamin A (retinol), in ethanol under UVA light irradiation was studied. The resulting photodecomposition products were separated by reversed-phase HPLC and identified by spectral analysis and comparison with the chromatographic and spectral properties of synthetically prepared standards. The identified products include 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), palmitic acid, ethyl palmitate, and four tentatively assigned cis and trans isomeric 15-ethoxy-ARs. AR was formed as a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Z-cis-AR with all-trans-AR predominating. 5,6-Epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP were also formed from reaction of RP with alkylperoxy radicals generated by thermal decomposition of 2,2'-azobis(2,4-dimethylvaleronitrile). Formation of these photodecomposition products was inhibited in the presence of sodium azide (NaN3), a free radical inhibitor. These results suggest that formation of 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP from photoirradiation of RP is mediated by a light-initiated free radical chain reaction. AR and the isomeric 11-ethoxy-ARs were not formed from reaction of RP with alkylperoxy radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile), and their formation was not inhibited when NaN3 was present during the photoirradiation of RP. We propose that these products were formed through an ionic photodissociation mechanism, which is similar to the reported formation of AR through ionic photodissociation of retinyl acetate. RP and all its identified photodecomposition products described above (i) were not mutagenic in Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 in the presence and absence of S9 activation enzymes, (ii) were not photomutagenic in Salmonella typhimurium TA102 upon UVA irradiation, and (iii) did not bind with calf thymus DNA in the presence of microsomal metabolizing enzymes. These results suggest that RP and its decomposition products are not genotoxic; however, photoirradiation of RP, 5,6-epoxy-RP, and AR with UVA light in the presence of methyl linoleate resulted in lipid peroxide (methyl linoleate hydroperoxides) formation. The lipid peroxide formation was inhibited by dithiothreitol (DTT) (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), and superoxide dismutase (SOD) (superoxide scavenger) but was enhanced by the presence of deuterium oxide (D2O) (enhancement of singlet oxygen lifetime). These results suggest that photoirradiation of RP, 5,6-epoxy-RP, and AR by UVA light generated reactive oxygen species resulting in lipid (methyl linoleate) peroxidation.

Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin

AbstractThe reaction mechanism of chitosan, bovine serum albumin (BSA), and gelatin with genipin (a natural crosslinking reagent) was examined with infrared, ultraviolet–visible, and 13C NMR spectroscopies; protein‐transfer reaction mass spectrometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two reactions that proceeded at different rates led to the formation of crosslinks between primary amine groups. The fastest reaction to occur was a nucleophilic attack on genipin by a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester group possessed by genipin to form a secondary amide link with chitosan, BSA, or gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second‐order gelation kinetics resulting from an irreversible crosslinking process, but was complicated by the oxygen radical‐induced polymerization of genipin that caused the gels to assume a blue color in the presence of air. The lower elastic modulus attained after a given time during crosslinking of the globular protein BSA as compared to the coiled protein gelatin, despite possessing more crosslinkable basic residues, demonstrated the importance of protein secondary and tertiary structures in determining the availability of sites for crosslinking with genipin in protein systems. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3941–3953, 2003

Mitochondrial permeability transition induced by chemically generated singlet oxygen.

Pure singlet molecular oxygen (1O2) generated by thermal decomposition of the 3,3'-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inhibited respiration of isolated rat liver mitochondria supported by NADH-linked substrates or succinate, but not by N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbate. Under the latter conditions, mitochondria treated with 2.7 mM NDPO2 exhibited a decrease in transmembrane potential (deltapsi) in manner dependent on NDPO2 exposure time. This process was sensitive to the mitochondrial permeability transition inhibitors EGTA, dithiothreitol, ADP, and cyclosporin A. The presence of deuterium oxide (D2O), that increases 1O2 lifetime, significantly enhanced NDPO2-promoted mitochondrial pereabilization. In addition, NDPO2-induced mitochondrial permeabilization was accompanied by DTT or ADP-sensitive membrane protein thiol oxidation. Taken together, these results provide evidence that mitochondrial permeability transition induced by chemically generated singlet oxygen is mediated by the oxidation of membrane protein thiols.

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Oxidation of Hypotaurine to Taurine with Photochemically Generated Singlet Oxygen: The Effect of Azide

Hypotaurine is oxidized to taurine by singlet oxygen (1O2) generated with methylene blue used as a photosensitizer. The oxidation rate increases in the presence of deuterium oxide as expected for the involvement of1O2. Addition of the1O2quencher azide also produced an activating effect in contrast with the expected inhibition. Azidyl radicals produced by the oxidation of azide by the horseradish peroxidase/hydrogen peroxide system stimulate the oxidation of the added hypotaurine. It is concluded that azide competes with hypotaurine for1O2generating the azidyl radical which is a strong one-electron oxidant transfer of the radical to hypotaurine. The hypotaurine radical is then converted into taurine, possibly through the disulfone intermediate. Formation of the sulfonic hydroperoxide is the possible intermediate in the absence of azide. The finding that the azidyl radical efficiently oxidizes hypotaurine to its metabolic product taurine raises the expectation of hypotaurine being a valuable scavenger of endogenous and exogenous radicals.

Quantification of singlet oxygen from hematoporphyrin derivative by electron spin resonance

The mechanism of the generation and the quantitative analysis of singlet oxygen ( 1O 2) formed by the exposure of a hematoporphyrin derivative (HpD) to light was re-evaluated by electron spin resonance (ESR) combined with 2,2,6,6,-tetramethyl-4-piperidine (TMPD). The change from TMPD to 2,2,6,6,-tetramethyl-4-piperidine-N-oxide (TAN) has been reported to depend on singlet oxygen. However,we confirmed that this reagent also reacts with Superoxide anion (O 2) and hydroxyl radicals (OH). Therefore, the reactions between TMPD and 1O 2, O 2 and OH were re-examined using a kinetic approach. We found that the generatiation of TAN was proportional to the concentration of TMPD and HpD, as well as to the duration and strength of the illumination. The generation of TAN was enhanced in the presence of deuterium oxide. The generation of TAN was not inhibited by dimethyl-sulfoxide (DMSO) or Superoxide dismutase (SOD). The reaction rate between TMPD and 1O 2 was determined to be 5.0 × 10 −7 M −1min −1. The generation of 1O 2 from HpD was 2.7 ×10 −7Mmin −1 under our conditions. The competitive reaction observed between 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and TMPD for O 2- or OH shows that TMPD reacts with both forms of active oxygen, but gave no ESR signal. The second-order reaction rate constant of TMPD between O 2 and OH was calculated as 73M −1S −1 and 1.5 × 10 9M −1S −1, respectively. The photochemical generation of 1O 2 from methylene blue, another sensitzer, was also demonstrated by this method.These results show that ESR signal of TAN can be used for the highly selective monitoring of 1O 2.

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.