Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (¹O₂) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of ¹O₂. In addition, the generation of ¹O₂ was verified further in the presence of D₂O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN₃). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of ¹O₂ by PBPs are as follows: APC > C-PC > R-PE.     

Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant

α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l⁻¹ for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH₂PO₄/Na₂HPO₄ buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity.     

Conversion of 2,5-dimethylfuran to 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran,a true 1O2-derived reaction in aqueous 1O2 generating systems

It has been studied whether 2,5-dimethylfuran (DMF) is a specific ¹O₂ trapping agent in aqueous system. The exposure of DMF to aqueous ¹O₂ generating system (Rose Bengal photooxygenation system) gave 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran (a hydrated form of endoperoxide, ¹O₂-derived reaction product) and cis-diacetylethylene (cis-DAE), while the bromine-catalyzed autoxidation of DMF afforded only trans-DAE. In Fenton system (·OH generating system) DMF was converted in the main to cis-DAE, but not to the hydrated form of endoperoxide. The exposure of DMF to acetaldehyde-xanthine oxidase system failed to detect the hydrated form of endoperoxide, but chiefly yielded a non-specific oxidation product, cis-DAE.     

Diminution of singlet oxygen-induced DNA damage by curcmin and related antioxidants

Curcumin, the natural antioxidant from turmeric, an Indian spice, and its derivatives have significant abilities to protect plasmid pBR322 against single-strand breaks induced by singlet oxygen (¹O₂), a reactive oxygen species with potential genotoxic/mutagenic properties. ¹O₂ was generated at 37°C in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate (NDPO₂). Among the compounds, tested, curcumin was the most effective inhibitor of DNA damage followed by desmethoxycurcumin, bisdesmethoxycurcumin and other derivatives. The observed antioxidant activity was both time-and concentration-dependent. The protectice ability of curcumin was higher than that of the well-known biological antioxidants lipoate, α-tocopherol and β-carotene. However, the highest protective ability with saturating concentrations of curcumin did not exceed 50%. The ability of curcumin and its derivatives to protect DNA against ¹O₂ seems to be related to their structures and may at least partly explain the therapeutic and other beneficial effects of these compounds including anticarcinogenic and antimutagenic properties.     

Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion

Abstract

Here, we determined the electron spin resonance (ESR) spectra of standard reaction mixtures (I) containing 25 μM flavin mononucleotide (FMN), 0.018% tea tree (Melaleuca alternifolia) oil, 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and 1.0 mM FeSO₄(NH₄)₂SO₄ irradiated with 436 nm visible light (7.8 J/cm²). Prominent ESR signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) were detected, suggesting that free radicals form in the standard reaction. In order to know whether singlet oxygen (¹O₂) is involved in the radical formation or not, ESR measurement was performed for the standard D₂O reaction mixture (I) which contained 25 μM FMN, 0.0036% tea tree oil, 1.9 M acetonitrile-d3, 20 mM phosphate buffer (pH 7.4), 0.1 M 4-POBN and 1.0 mM FeSO₄ in D₂O. The ESR peak height of the standard D₂O reaction increased to 169 ± 24% of the control. Thus, ¹O₂ seems to be involved in the formation of the radicals because D₂O increases the lifetime of singlet oxygen. High-performance liquid chromatography-ESR-mass spectrometry analyses detected 1-methylethyl and methyl radicals in the standard reaction. The radicals appear to form through the reaction of ferrous ion with α-terpinene endoperoxide (ascaridole), which generated from the reaction of α-terpinene with ¹O₂. The 1-methylethyl and methyl radicals may exert a pro-oxidant effect under these conditions.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Effects of gassing,pH, quenching agents and photodynamically active compounds on photobleaching and photoinhibition of the cyanobacterium Anabaena variabilis

The findings presented in this paper support the suggestion that in the cyanobacterium Anabaena variabilis photobleaching is the result of an increased intracellular level of singlet molecular oxygen, whereas photoinhibition is controlled by a different molecular mechanism. Photobleaching of Anabaena trichomes can be prevented effectively by gassing with argon, nitrogen and carbon dioxide as well as by treatment with the ¹O₂ quenchers sodium azide and crocetin, and finally, with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, photodynamically active compounds, capable of ¹O₂ generation, increase photobleaching drastically. Thus, photobleaching is probably caused by singlet molecular oxygen. Photoinhibition studied with the aid of the fluorescence induction was not prevented by most of the treatments which prevent photobleaching. Therefore, different control mechanisms have to be assumed for this process.Abbreviations DABCO 1,4-diazabicyclo(2,2,2)octane - DBMIB dibromothymoquinone = (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - C-PC C-phycocyanin - Chl a chlorophyll a - LFE low fluence rate exposure - HFE high fluence rate exposure     

Oxygenation reactions of prostaglandins endoperoxide synthase and soybean lipoxygenase. Surprising stoichiometry in the formation of hydroperoxy and hydroxy derivatives of cis,cis-eicosa-11,14-dienoic acid

The stoichiometry of the oxygenation reaction of cis,cis-eicosa-11,14-dienoic acid catalyzed by prostaglandin endoperoxide synthase and soybean lipoxygenase has been investigated by using steady-state initial rate measurements. The rate of product formation (conjugated diene hydroperoxy and hydroxy derivatives) was followed spectrophotometrically at 235 nm, and the rate of oxygen consumption was measured polarographically. The ratio of the two rates, d[conjugated diene]/-d[O₂], is 2/1 for the prostaglandin endoperoxide synthase catalyzed reaction and 1/1 for the lipoxygenase reaction. The 2/1 ratio can be explained by two interrelated routes, each of which results in formation of the conjugated diene hydroxy derivative of the acid. One route, initiated by hydrogen atom abstraction from the acid by Compound I, results in formation of the conjugated diene hydroperoxy derivative. The latter is converted to the hydroxy derivative by regenerating Compound I from the native enzyme. The other route involves direct oxygen atom insertion into the acid by the tyrosyl radical form of Compound I. The decrease in absorbance at 235 nm obtained in the presteady-state phase suggests that during the initial contact of hydroperoxide and enzyme an epoxy-hydroxy fatty acid-enzyme complex may be formed.     

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by singlet oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, back mutation from arg₅₆^? to arg⁺ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (¹O₂) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D₂O and in H₂O media (pH 7.0) and quenching of ¹O₂ with 1,4-diazabicyclo[2.2.2]octane (DABCO).When photodynamy was performed with the indicator cells suspended in D₂O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H₂O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D₂O itself was not mutagenic under the present experimental conditions. Addition of DABCO in concentrations of 0.1–10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions.From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that ¹O₂ generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.     

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 () 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 permeabilization. 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.     

Possible Mechanism of Oxygen Activation in Linoleate Peroxidation by Fusarium Lipoxygenase

The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxygenase, was studied. The enzyme did not require any cofactors, such as H₂O₂, however, both superoxide dismutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H₂O₂ caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O₂? and H₂O₂ are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase. This peroxidation reaction was also prevented by scavengers of singlet oxygen (¹O₂), but not by scavengers of hydroxy 1 radical (OH). Generation of O₂? in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide formation was greater in the D₂O than in the H₂O buffer system. These results suggest that the Haber–Weiss reaction (O₂^?+H₂O₂→OH^?+OH^·+¹O₂) is taking part in linoleate peroxidation by Fusarium lipoxygenase, and the ¹O₂ evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other heme-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.     

Reactivities of diphenylfuran (a singlet oxygen trap) with singlet oxygen and hydroxyl radical in aqueous systems.

It has been studied whether 2,5-diphenylfuran is a specific singlet oxygen trap in aqueous systems. With certain ¹O₂ generating systems (Rose Bengal photooxygenation and NaOClH₂O₂ systems) and·OH generating systems (Fenton's reagent and acetaldehyde-xanthine oxidase system), diphenylfuran was chiefly converted in all cases to cis-dibenzoyl-ethylene, but not to trans-dibenzoylethylene. Low but detectable conversion of diphenylfuran to a hydroperoxide, probably a distinct ¹O₂-derived reaction in aqueous media, was found only in the Rose Bengal photooxygenation system.     

Ultraviolet-B inhibition of motility in green and dark bleachedEuglena gracilis

The motility of green and dark bleachedEuglena gracilis was studied under artificial and solar UV-B radiation. The percentage of motile cells in the population was drastically impaired after exposure to unfiltered sunlight for only a few hours. Dark bleached cells were even more affected than green organisms. The effect is caused mainly by the solar UV-B component, since filtering the sunlight by either a layer of ozone or a UV-B-absorbing filter substantially increased the survival rate. Addition ofp-quinone, a scavenger of free radicals produced in a type I photodynamic reaction, did not relieve the UV-B effects, but was cytotoxic at higher concentrations. Likewise, 1,4-diazobicyclo[2,2,2]octane and imidazole, which quench singlet oxygen (¹O₂) generated in a type II photodynamic reaction, did not prolong the survival in UV-B irradiation. D₂O, which, in contrast, prolongs the lifetime of¹O₂, is tolerated by the cells but does not aggravate the UV-B inhibition. Thus, photodynamic processes of both type I and II can be ruled out as possible mechanisms of UV-B inhibition of motility inEuglena gracilis.     

Stability of Glucose 6-Phosphate Dehydrogenase Complexed with Its Substrate or Cofactor in Aqueous and Micellar Environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), or cofactor, NADP⁺, has been studied within the range 20–40°C in three media: (a) 0.04 M NaOH–glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, k _in(s^–1). In the case of G6PDH–NADP⁺complexes, the values of k _inwere independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of k _infor the complex G6PDH–GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of k _incorresponded to the degree of hydration W ₀= 16.7; at W ₀> 16.7 and W ₀< 16.7, k _inincreased. Within the range 20–40°C, the values of k _inmeasured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of k _inwere characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6°C to 40°C. In all media studied, NADP⁺complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH–NADP⁺melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Integrated two‐liquid phase bioconversion and product‐recovery processes for the oxidation of alkanes: Process design and economic evaluation

Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two‐liquid phase bioreactor systems. In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non‐water soluble substrates. The apolar products typically accumulate in the emulsified apolar phase. We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two‐liquid phase media. Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1‐alkanols from n‐alkanes on a 10 kiloton/yr scale, taking the conversion of n‐octane to 1‐octanol as a model system. Here, we describe overall designs of fed‐batch and continuous‐fermentation processes for the oxidation of octane to 1‐octanol by Pseudomonas oleovorans, and we discuss the economics of these processes. In both systems the two‐liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n‐octane is dissolved, while the cells are present in the aqueous phase. In one system, multiple‐batch fermentations are followed by continuous processing of the product from the separated apolar phase. The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product. Fewer fermentors were required and a higher space‐time‐yield was possible for production of 1‐octanol in a continuous process. The overall performance of each of these two systems has been modeled with Aspen software. Investment and operating costs were estimated with input from equipment manufacturers and bulk‐material suppliers. Based on this study, the production cost of 1‐octanol is about 7 US$kg⁻¹ when produced in the fed‐batch process, and 8 US$kg⁻¹ when produced continuously. The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed‐batch process and slightly higher upstream costs for continuous fermentation. The largest cost contribution was due to variable production costs, mainly resulting from media costs. The organisms used in these systems are P. putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further. Hence, such cells need a second carbon source, which in these systems is glucose. Although the continuous process is about 10% more expensive than the fed‐batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed‐batch fermentation by decreasing the dilution rate while maintaining near constant productivity. Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 84: 459–477, 1999.     

Role of reactive oxygen species in cardiac preconditioning: Study with photoactivated rose bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.