Irene E. Kochevar

Spatially resolved cellular responses to singlet oxygen.

Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 μs in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only ?220 nm during three lifetimes (?5%1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo‐oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 1O2 occur in cells.Abstract Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 μs in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only ∼220 nm during three lifetimes (∼5% 1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo-oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 1O2 occur in cells.

[2] Photosensitized production of singlet oxygen

Photosensitization is a simple and controllable method for the generation of singlet oxygen in solution and in cells. Methods are described for determining the yield of singlet oxygen in solution, for measurement of the rate of reaction between singlet oxygen and a substrate, and for comparing the effectiveness of singlet oxygen generated by different photosensitizers in cells. These quantitative measurements can lead to better understanding of the interaction of singlet oxygen with biomolecules.

Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures

The non-invasive determination of deep tissue three dimensional structure and biochemistry is the ultimate goal of optical biopsy. Two-photon microscopy has been shown to be a particularly promising approach. The use of infrared radiation in two-photon microscopy is critical for deep tissue imaging since tissue absorption and scattering coefficients for infrared light are much lower than for shorter wavelengths. Equally important, tissue photodamage is localized to the focal region where fluorescence excitation occurs. This report demonstrates that, by means of high resolution two-photon microscopy, skin and subcutaneous tissue structures can be imaged utilizing their endogenous fluorescence. From a freshly prepared tissue punch of a mouse ear, we were able to 3D resolve both the living and cornified keratinocytes in the epidermis, the collagen/elastin fibers in the dermal layer and the cartilage in the subcutaneous layer. The ability to non-invasively acquire 3D structures of these tissue components may find application in areas such as non-invasive diagnosis of skin cancer and the study of wound healing processes.

Electron Transfer Quenching of the Rose Bengal Triplet State

Abstract— The potential for electron transfer quenching of rose bengal triplet (3RB2‐) to compete with energy transfer quenching by oxygen was evaluated. Rate constants for oxidative and reductive quenching were measured in buffered aqueous solution, acetonitrile and in small unilamellar liposomes using laser flash photolysis. Biologically relevant quenchers were used that varied widely in structure, reduction potential and charge. Radical ion yields (Øi) were measured by monitoring the absorption of the rose bengal semireduced (RB*3‐) and semioxidized (RB*‐) radicals. The results in solution were analyzed as a function of the free energy for electron transfer (δG) calculated using the Weller equation including electrostatic terms. Exothermic oxidative quenching was about 10‐fold faster than exothermic reductive quenching in aqueous solution. The quenching rate constants decreased as δG approached zero in both aqueous and acetonitrile solution. Exceptions to these generalizations were observed that could be rationalized by specific steric or electrostatic effects or by a change in mechanism. The results suggest that electron transfer reactions with some potential quenchers in cells could compete with formation of singlet oxygen [O2(1δg)]. Values of Øi were generally greater for reductive quenching and, for oxidative quenching, greater in acetonitrile than in buffer. Electron transfer quenching of 3RB2‐ in liposomes, below the phase transition temperature was slower than in solution for both lipid‐soluble and water‐soluble quenchers indicating that these reactions may not compete with formation of O2(1δg) during cell photosensitization.

Chlorpromazine and protriptyline phototoxicity: photosensitized, oxygen independent red cell hemolysis.

Abstract—Red blood cell lysis photosensitized by chlorpromazine and protriptyline was investigated. Oxygen independent sensitized photohemolysis was observed with both compounds. When chlorpromazine and protriptyline were irradiated in the absence of red cells, both formed photoproducts which lysed red cells. These results indicate that chlorpromazine and protriptyline represent a new mechanistic class of phototoxic compounds which photosensitize membrane disruption by oxygen independent processes.

Collagen Cross-Linking Using Rose Bengal and Green Light to Increase Corneal Stiffness

PURPOSE Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application. METHODS Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment. RESULTS RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability. CONCLUSIONS RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

Singlet Oxygen, but not Oxidizing Radicals, Induces Apoptosis in HL-60 Cells¶

Abstract Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.

MECHANISMS OF DRUG PHOTOSENSITIZATION

Drug photosensitization includes phototoxic and photoallergic reactions with phototoxicity being the much more common. Phototoxicity usually appears as a sunburnlike reaction within minutes to a few days of initial exposure to the drug and the appropriate wavelengths of light. The reaction can be demonstrated in most exposed individuals and is not an immune response. Photoallergy usually appears as an eczematous dermatitis which begins a week or more after initial exposure to the drug and light. Only a few of the exposed individuals show this immune response. This review focuses on the articles published in 1985 and 1986 on mechanisms of adverse photosensitivity reactions to drugs. Photosensitization may also be beneficial such as in the photochemotherapies using psoralens and porphyrins. Photosensitization related to these therapeutic uses has been reviewed in this Journal and others recently, and is not covered in this review (Moan, 1986; Spikes, 1986; Kessel, 1984; Pathak, 1984). Photosensitization by plants, perfume ingredients, and industrial compounds share many of the mechanistic patterns as photosensitization by drugs, but these are not reviewed. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been the focus of recent phototoxicity research. In addition to the articles cited below on the mechanisms of phototoxicity to specific NSAIDs, three general articles have been published. Results of in vitro phototoxicity assays were reviewed and indicated that many of these agents were phototoxic including azapropazone, benoxaprofen, carprofen, diclofenac, diflunisal, ibuprofen ketoprofen, naproxen and tiaprofenic acid (Przybilla et al., 1986). Fourteen NSAIDs were tested in an in vivo assay for phototoxicity (Ljunggren and Lundberg, 1986). Of these, tiaprofenic acid, carprofen, benoxaprofen, and naproxen were phototoxic. An in vitro assay also indicated that propionic, acid-derived NSAIDs were more likely to be phototoxic than other classes tested (Ljunggren, 1985).

Photoallergic contact dermatitis to musk ambrette: Clinical report of two patients with persistent light reactor patterns

Two male patients with photoallergic contact dermatitis to musk ambrette, 2-methoxy-3,5-dinitro-4-methyl-t-butylbenzene, are reported. Musk ambrette is a synthetic fragrance material commonly used in foods and cosmetics. The clinical distribution of lesions presented by these patients strongly suggested an adverse reaction to light. Over a period of several years no etiology had been determined in these cases despite extensive testing and hospitalization. Photopatch tests to musk ambrette were positive. No other etiology for photosensitivity was found. This report emphasizes that exposure to household or cosmetic products represents a potential source of contact photosensitivity. Patients presenting with dermatoses of unknown origin confined to the light-exposed areas should have fragrances considered as possible etiologic agents.

Low level light effects on inflammatory cytokine production by rheumatoid arthritis synoviocytes

Low level light therapy (LLLT) is being evaluated for treating chronic and acute pain associated with rheumatoid arthritis (RA) and other inflammatory diseases. The mechanisms underlying the effectiveness of LLLT for pain relief in RA are not clear. The objectives of this study were to determine whether LLLT decreased production of pro‐inflammatory cytokines by cells from RA joints, and, if so, to identify cellular mechanisms.