Effects of low temperature on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis.

MAIN CONCLUSIONS: Low temperature decreases PSII damage in vivo, confirming earlier in vitro results. Susceptibility to photoinhibition differs among Arabidopsis accessions and moderately decreases after 2-week cold-treatment. Flavonols may alleviate photoinhibition. The rate of light-induced inactivation of photosystem II (PSII) at 22 and 4 °C was measured from natural accessions of Arabidopsis thaliana (Rschew, Tenela, Columbia-0, Coimbra) grown under optimal conditions (21 °C), and at 4 °C from plants shifted to 4 °C for 2 weeks. Measurements were done in the absence and presence of lincomycin (to block repair). PSII activity was assayed with the chlorophyll a fluorescence parameter Fv/Fm and with light-saturated rate of oxygen evolution using a quinone acceptor. When grown at 21 °C, Rschew was the most tolerant to photoinhibition and Coimbra the least. Damage to PSII, judged from fitting the decrease in oxygen evolution or Fv/Fm to a first-order equation, proceeded more slowly or equally at 4 than at 22 °C. The 2-week cold-treatment decreased photoinhibition at 4 °C consistently in Columbia-0 and Coimbra, whereas in Rschew and Tenela the results depended on the method used to assay photoinhibition. The rate of singlet oxygen production by isolated thylakoid membranes, measured with histidine, stayed the same or slightly decreased with decreasing temperature. On the other hand, measurements of singlet oxygen from leaves with Singlet Oxygen Sensor Green suggest that in vivo more singlet oxygen is produced at 4 °C. Under high light, the PSII electron acceptor QA was more reduced at 4 than at 22 °C. Singlet oxygen production, in vitro or in vivo, did not decrease due to the cold-treatment. Epidermal flavonols increased during the cold-treatment and, in Columbia-0 and Coimbra, the amount correlated with photoinhibition tolerance.

Self-quenching synthesis of coordination polymer pre-drug nanoparticles for selective photodynamic therapy.

The design and preparation of a photoactive coordination polymer nanoplatform with tumor-related stimuli-activatability and biodegradability is highly desirable for achieving highly precise photodynamic therapy (PDT). Herein, novel "pre-photodynamic" nanoparticles (Fe-IBDP NPs) with a tumor microenvironment (TME)-activatable PDT and good biodegradability were synthesized by carrying out facile coordination assembly of an IBDP photosensitizer with an Fe3+ quenching agent. After being taken up by cancer cells, our "inactive" Fe-IBDP NPs were activated by the TME and as a result decomposed and released the photoactive carboxyl-functionalized diiodo-substituted BODIPY (IBDP) photosensitizer, which generated cytotoxic singlet oxygen (1O2) under light irradiation. By contrast, these NPs showed relatively low cytotoxicity in normal cells. This work also provided a feasible method for preparing the next generation of photoactive nanomedicines for use in precise phototherapy.

Calcium carbonate-methylene blue nanohybrids for photodynamic therapy and ultrasound imaging.

Photodynamic therapy plays an important role in cancer treatment. In this work, methylene blue (MB)-embedded calcium carbonate nanorods (CaCO3-MB NRs) have been synthesized for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO3-MB NRs can be controlled by modulating the concentration of Na2CO3 aqueous solution. The generation of effective reactive oxygen species (ROS) were confirmed by 1,3-diphenylisobenzofuran (DPBF) probe. Both photodynamic therapy performance and echogenic performance of CaCO3-MB NRs were investigated to confirm the feasibility of CaCO3-MB nanohybrids for ultrasound image-guided photodynamic therapy.

Direct 1O2 optical excitation: A tool for redox biology.

Molecular oxygen (O2) displays very interesting properties. Its first excited state, commonly known as singlet oxygen (1O2), is one of the so-called Reactive Oxygen Species (ROS). It has been implicated in many redox processes in biological systems. For many decades its role has been that of a deleterious chemical species, although very positive clinical applications in the Photodynamic Therapy of cancer (PDT) have been reported. More recently, many ROS, and also 1O2, are in the spotlight because of their role in physiological signaling, like cell proliferation or tissue regeneration. However, there are methodological shortcomings to properly assess the role of 1O2 in redox biology with classical generation procedures. In this review the direct optical excitation of O2 to produce 1O2 will be introduced, in order to present its main advantages and drawbacks for biological studies. This photonic approach can provide with many interesting possibilities to understand and put to use ROS in redox signaling and in the biomedical field.

Singlet oxygen production by combining erythrosine and halogen light for photodynamic inactivation of Streptococcus mutans.

BACKGROUND: Photodynamic inactivation of microorganisms is based on a photosensitizing substance which, in the presence of light and molecular oxygen, produces singlet oxygen, a toxic agent to microorganisms and tumor cells. This study aimed to evaluate singlet oxygen quantum yield of erythrosine solutions illuminated with a halogen light source in comparison to a LED array (control), and the photodynamic effect of erythrosine dye in association with the halogen light source on Streptococcus mutans. METHODS: Singlet oxygen quantum yield of erythrosine solutions was quantified using uric acid as a chemical-probe in an aqueous solution. The in vitro effect of the photodynamic antimicrobial activity of erythrosine in association with the halogen photopolimerizing light on Streptococcus mutans (UA 159) was assessed during one minute. Bacterial cultures treated with erythrosine alone served as negative control. RESULTS: Singlet oxygen with 24% and 2.8% degradation of uric acid in one minute and a quantum yield of 0.59 and 0.63 was obtained for the erythrosine samples illuminated with the halogen light and the LED array, respectively. The bacterial cultures with erythrosine illuminated with the halogen light presented a decreased number of CFU mL(-1) in comparison with the negative control, with minimal inhibitory concentrations between 0.312 and 0.156mgmL(-1). CONCLUSIONS: The photodynamic response of erythrosine induced by the halogen light was capable of killing S. mutans. Clinical trials should be conducted to better ascertain the use of erythrosine in association with halogen light source for the treatment of dental caries.

Quinones as photosensitizer for photodynamic therapy: ROS generation, mechanism and detection methods.

Photodynamic therapy (PDT) is based on the dye-sensitized photooxidation of biological matter in the target tissue, and utilizes light activated drugs for the treatment of a wide variety of malignancies. Quinones and porphyrins moiety are available naturally and involved in the biological process. Quinone metabolites perform a variety of key functions in plants which includes pathogen protection, oxidative phosphorylation, and redox signaling. Quinones and porphyrin are biologically accessible and will not create any allergic effects. In the field of photodynamic therapy, porphyrin derivatives are widely used, because it absorb in the photodynamic therapy window region (600-900 nm). Hence, researchers synthesize drugs based on porphyrin structure. Benzoquinone and its simple polycyclic derivatives such as naphthaquinone and anthraquinones absorb at lower wavelength region (300-400 nm), which is lower than porphyrin. Hence they are not involved in PDT studies. However, higher polycyclic quinones absorb in the photodynamic therapy window region (600-900 nm), because of its conjugation and can be used as PDT agents. Redox cycling has been proposed as a possible mechanism of action for many quinone species. Quinones are involved in the photodynamic as well as enzymatic generation of reactive oxygen species (ROS). Generations of ROS may be measured by optical, phosphorescence and EPR methods. The photodynamically generated ROS are also involved in many biological events. The photo-induced DNA cleavage by quinones correlates with the ROS generating efficiencies of the quinones. In this review basic reactions involving photodynamic generation of ROS by quinones and their biological applications were discussed.

Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms.

BACKGROUND DATA: Methylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms. METHODS: Photoactivated formulations were prepared by diluting the Ps (250 µM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log10) data were evaluated by two-way ANOVA and post-hoc Tukey's tests. RESULTS: In all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P=0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control. CONCLUSIONS: MB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.

LIGHT-SABRE enables efficient in-magnet catalytic hyperpolarization.

Nuclear spin hyperpolarization overcomes the sensitivity limitations of traditional NMR and MRI, but the most general method demonstrated to date (dynamic nuclear polarization) has significant limitations in scalability, cost, and complex apparatus design. As an alternative, signal amplification by reversible exchange (SABRE) of parahydrogen on transition metal catalysts can hyperpolarize a variety of substrates, but to date this scheme has required transfer of the sample to low magnetic field or very strong RF irradiation. Here we demonstrate "Low-Irradiation Generation of High Tesla-SABRE" (LIGHT-SABRE) which works with simple pulse sequences and low power deposition; it should be usable at any magnetic field and for hyperpolarization of many different nuclei. This approach could drastically reduce the cost and complexity of producing hyperpolarized molecules.

Photostabilization of endogenous porphyrins: excited state quenching by fused ring cyanoacrylates.

Endogenous chromophores in human skin, when exposed to sunlight, generate harmful reactive oxygen species (ROS). Protoporphyrin IX (PpIX) is one of the common chromophores in human tissue. A series of aromatic cyanoacrylates were tested as quenchers of excited singlet and triplet states of PpIX. While the diaryl cyanoacrylate () did not quench excited singlet or triplet states of PpIX, some cyanoacrylates with fused aromatic rings showed excited singlet state quenching rate constants as high as 5 × 10(9) M(-1) s(-1) (acetonitrile solution). In addition, one of the fused ring cyanoacrylates () quenches PpIX triplet states with a rate constant of 3 × 10(9) M(-1) s(-1). The observed quenching rate constants correlated well with the suppression of singlet oxygen generation from PpIX under visible light exposure in the presence of dissolved oxygen. This photostabilization of endogenous chromophores can prevent or reduce ROS generation and perhaps constitute a new approach to mitigating cutaneous oxidative stress.

Exposure of vitamins to UVB and UVA radiation generates singlet oxygen.

Deleterious effects of UV radiation in tissue are usually attributed to different mechanisms. Absorption of UVB radiation in cell constituents like DNA causes photochemical reactions. Absorption of UVA radiation in endogenous photosensitizers like vitamins generates singlet oxygen via photosensitized reactions. We investigated two further mechanisms that might be involved in UV mediated cell tissue damage. Firstly, UVB radiation and vitamins also generate singlet oxygen. Secondly, UVB radiation may change the chemical structure of vitamins that may change the role of such endogenous photosensitizers in UVA mediated mechanisms. Vitamins were irradiated in solution using monochromatic UVB (308 nm) or UVA (330, 355, or 370 nm) radiation. Singlet oxygen was directly detected and quantified by its luminescence at 1270 nm. All investigated molecules generated singlet oxygen with a quantum yield ranging from 0.007 (vitamin D3) to 0.64 (nicotinamide) independent of the excitation wavelength. Moreover, pre-irradiation of vitamins with UVB changed their absorption in the UVB and UVA spectral range. Subsequently, molecules such as vitamin E and vitamin K1, which normally exhibit no singlet oxygen generation in the UVA, now produce singlet oxygen when exposed to UVA at 355 nm. This interplay of different UV sources is inevitable when applying serial or parallel irradiation with UVA and UVB in experiments in vitro. These results should be of particular importance for parallel irradiation with UVA and UVB in vivo, e.g. when exposing the skin to solar radiation.

[measurement of optical density in infrared absorption maxima of oxygen molecules based on their photochemical activity upon direct laser excitation].

Generation of singlet oxygen upon excitation of oxygen molecules by infrared diode lasers has been studied in organic media (carbon tetrachloride and acetone) saturated by air under normal pressure and temperature. A new approach to analysis of the experimental data has been developed taking into account a degree of overlapping of the spectral bands of oxygen and laser radiation. Optical density, molar absorption coefficient and the cross section of light absorption were determined for the main absorption maxima of O2 at 765 and 1273 nm. The results are compared with the data of previous studies. A significance of the obtained results for elucidation of photophysics and photochemistry of oxygen molecules and investigation of biological action of laser radiation is discussed.

Singlet oxygen mediated DNA damage induced phototoxicity by ketoprofen resulting in mitochondrial depolarization and lysosomal destabilization.

Ketoprofen (KP) is a widely used nonsteroidal anti-inflammatory drug for the treatment of osteoarthritis and various rheumatic diseases. Currently, KP is applied topically on skin as gel to treat symptoms of pain and inflammation. We have studied the photomodification of KP under natural environmental conditions. KP generates reactive oxygen species (ROS) like ¹O2 through Type-II photodynamic reaction. ¹O2 mediated 2'-dGuO photodegradation, single and double strand breakage were significantly induced by photosensitized KP under sunlight/UV-R exposure. Significant intracellular ROS generation was measured through DCF-DA fluorescence. Linoleic acid photoperoxidation and role of ¹O2 were substantiated by using specific quencher like sodium azide. KP induced cell cycle arrest in G2/M phase and cell death through MTT assay. We found apoptosis as the pattern of cell death which was confirmed through caspase-3 activation, cytochrome-c release from mitochondria, up-regulation of Bax protein and phosphatidylserine translocation. Our RT-PCR result strongly supports our view point of apoptotic cell death through up-regulation of p21 and pro-apoptotic Bax genes expression. Mitochondrial depolarization and lysosomal destabilization were also parallel to apoptotic process. Therefore, much attention should be paid to the topical application of KP and sunlight exposure in the light of skin related photosensitivity and cancers.

New insights to primary photodynamic effects--Singlet oxygen kinetics in living cells.

The kinetics of chemical singlet oxygen quencher consumption inside living cells during low dose illumination was revealed via time resolved singlet oxygen luminescence detection. Deviations of the measured data from the common theoretical model for (1)O(2) kinetics forced the authors to consider a one-dimensional diffusion model for description of the kinetics of singlet oxygen generated by membrane localized photosensitizers. Our observations reconcile seemingly contradictory reports presenting different values for the efficiency of singlet oxygen interaction with cells.

High-light induced singlet oxygen formation in cytochrome b(6)f complex from Bryopsis corticulans as detected by EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen ((1)O(2)*) in the intact and the Rieske-depleted cytochrome b(6)f complexes (Cyt b(6)f) from Bryopsis corticulans, as well as in the isolated Rieske Fe-S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b(6)f can be bleached by light-induced (1)O(2)*, and that the (1)O(2)* production can be promoted by D(2)O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, beta-carotene and glutathione. Under similar experimental conditions, (1)O(2)* was also detected in the Rieske-depleted Cyt b(6)f complex, but not in the isolated Rieske Fe-S protein. The results prove that Chl a cofactor, rather than Rieske Fe-S protein, is the specific site of (1)O(2)* formation, a conclusion which draws further support from the generation of (1)O(2)* with selective excitation of Chl a using monocolor red light.

Squalene as a target molecule in skin hyperpigmentation caused by singlet oxygen.

Based on our previous finding (Biochem. Biophys. Res. Commun., 223, 578-582, 1996) of singlet oxygen generation from coproporphyrin excreted on the skin surface from Propionibacterium acnes, we hypothesized that singlet oxygen formed in this way under UV exposure would promote peroxidation of skin surface lipids. We found that squalene was oxidized efficiently by singlet oxygen derived from coproporphyrin under UV exposure, and that the rate constant of squalene peroxidation by singlet oxygen was ten-fold higher than that of other skin surface lipids examined. The reaction was promoted more efficiently by UVA than by UVB. Furthermore, we found that topical application of squalene peroxide induced skin hyperpigmentation through increasing prostaglandin E(2) release from keratinocytes in guinea pigs. These results suggest that squalene peroxide formation by singlet oxygen plays a key role in photo-induced skin damage.

Excited state characteristics of 6-azauracil in acetonitrile: drastically different relaxation mechanism from uracil.

Excited-state dynamics of 6-azauracil (6-AU) and sensitized singlet oxygen formation in acetonitrile solution with UV irradiation were investigated for the first time. In the transient absorption measurement, the 248 nm laser photolysis gave a relatively intense absorption band at 320 nm (= 1100 +/- 100 dm(3) mol(-1) cm(-1)) and a broadband in the 500-700 nm region due to triplet 6-AU. The triplet 6-AU, decaying with the rate constant of (5.3 +/- 0.2) x 10(6) s(-1) in Ar saturated acetonitrile, was quenched by molecular oxygen with the rate constant of (2.5 +/- 0.1) x 10(9) dm(3) mol(-1) s(-1). The formation quantum yield of excited triplet 6-AU was estimated to be unity by acetone triplet sensitization and actinometry with benzophenone. The time-resolved thermal lensing signal of 6-AU was also observed by 248 nm laser excitation. In the presence of molecular oxygen, the sensitization from triplet 6-AU gave rise to formation of singlet oxygen O(2) ((1)Delta(g)) with a quantum yield of 0.63 +/- 0.03. Drastically different excited-state dynamics of aza-substituted uracil from normal uracil were clarified, and the mechanism for the enhancement of intersystem crossing by aza-substitution is discussed.

Optical detection of singlet oxygen produced by fatty acids and phospholipids under ultraviolet A irradiation.

Ultraviolet A (UVA) radiation has been known to generate reactive oxygen species, such as singlet oxygen, in skin, leading to the oxidation of lipids and proteins. This oxidation influences cellular metabolism and can trigger cellular signaling cascades, since cellular membranes and the stratum corneum contain a substantial amount of fatty acids and lipids. Using highly sensitive IR-photomultiplier technology, we investigated the generation of singlet oxygen by fatty acids and lipids. In combination with their oxidized products, the fatty acids or lipids produced singlet oxygen under UVA radiation at 355 nm that is directly shown by luminescence detection. Linolenic or arachidonic acid showed the strongest luminescence signals, followed by linoleic acid and docohexaenoic acid. The amount of singlet oxygen induced by lipids such as phosphatidylcholine was significantly higher compared to the corresponding fatty acids within phospholipids. This result indicates a synergistic process of oxygen radicals and singlet oxygen during irradiation. UVA radiation initiates singlet oxygen generation, which subsequently oxidizes other fatty acids that in turn produce additional singlet oxygen. This leads to an enhancement of UVA-induced damage of fatty acids and lipids, which must enhance the oxidative damages in cells.

Síntese e estudo das propriedades fotoinduzidas de derivados fenotiazínicos em sistemas biomiméticos / Synteses and study of photoinduced properties of phenothiazine derivatives on biomimetic systems

Neste trabalho são apresentados estudos do efeito de interfaces nas propriedades fotofísicas e fotoquímicas do azul de metileno (AM) e de derivados fenotiazínicos com o intuito de avaliar o potencial destes compostos como fotossensibilizadores (FS) em terapia fotodinâmica. As propriedades físico-químicas do AM foram estudadas em soluções de SDS e observou-se que a presença do AM em solução altera o equilíbrio entre as micelas de SDS, -diminuindo o valor da concentração micelar crítica de 7mmolL-1 para 70µmoIL-1. A presença das micelas em solução também interfere nas propriedades do AM. Em baixas concentrações de SDS há formação de dímeros de AM, constatados pelo aumento da absorbância em 580nm e diminuição da emissão de fluorescência. A caracterização das espécies transientes mostrou a existência de moléculas de azul de metileno no estado triplete (3AM) e de oxigênio singlete em soluções com altas concentrações de SDS e a formação de espécies radicalares do AM em baixas concentrações do tensoativo. Esta observação sugere que o mecanismo fotoquímico do AM é dependente da sua concentração local próxima de interfaces carregadas. As interações do AM e de alguns de seus derivados fenotiazínicos (tionina, azure A e azure B) com vesículas e com células...

Regulation of singlet oxygen generation using single-walled carbon nanotubes.

We have designed a novel photodynamic therapy (PDT) agent using protein binding aptamer, photosensitizer, and single-walled carbon nanotube (SWNT). The PDT is based on covalently linking a photosensitizer with an aptamer then wrapping onto the surface of SWNTs, such that the photosensitizer can only be activated by light upon target binding. We have chosen the human alpha-thrombin aptamer and covalently linked it with Chlorin e6 (Ce6), which is a second generation photosensitizer. Our results showed that SWNTs are great quenchers to singlet oxygen generation (SOG). In the presence of its target, the binding of target thrombin will disturb the DNA interaction with the SWNTs and cause the DNA aptamer to fall off the SWNT surface, resulting in the restoration of SOG. This study validated the potential of our design as a novel PDT agent with regulation by target molecules, enhanced specificity, and efficacy of therapeutic function, which directs the development of photodynamic therapy to be safer and more selective.