INTERACTION OF PHOTODYNAMICALLY INDUCED CELL KILLING and DARK CYTOTOXICITY OF RHODAMINE 123

Abstract— Loss of clonogenicity of Chinese hamster ovary (CHO) cells, murine L929 fibroblasts and human bladder carcinoma T24 cells caused by photodynamic treatment (PDT) with hematoporphyrin derivative (HPD) is synergistically enhanced by subsequent incubation with rhodamine 123 in the dark. For CHO and L929 cells this synergistic interaction can be explained by an increased uptake of rhodamine 123 as the result of the photodynamic treatment. With aluminum phthalocyanine (AIPc) as photosensitizer only additive effects were observed in the three cell lines. Incubation in the dark with rhodamine 123, followed by a photodynamic treatment with HPD, resulted in an antagonistic interaction with regard to loss of colony formation. With AIPc the combination of treatments resulted in an additive effect with L929 and T24 cells, whereas with CHO cells a slight antagonistic interaction was observed. An antagonistic effect was also observed in model experiments, treating histidine photodynamically with HPD and measuring oxygen consumption. A possible explanation of these results could be an interaction or complex formation of rhodamine 123 with HPD resulting in a diminished singlet oxygen production. With AIPc this does not take place.     

MEKC‐LIF analysis of rhodamine123 delivered by carbon nanotubes in K562 cells

Oxidized single‐walled carbon nanotubes (o‐SWNTs) were employed as the drug carriers to deliver the small molecules of Rhodamine123 (Rho123) into the K562 cells via physical adsorption. However, due to the fluorescence quenching of Rho123 on carbon nanotubes, the quantitative determination of Rho123 in cells is difficult. Based on the MEKC coupled with LIF detection, a quantitative approach was developed for the determination of Rho123 delivered into K562 cells by o‐SWNTs. Where the adsorbed Rho123 on o‐SWNTs could be desorbed by SDS in running buffer and be simultaneously separated with o‐SWNTs due to the differences of their electrophoretic mobility by applying the electric potential at the both ends of capillary. Using this approach, the intracellular uptakes of Rho123 in multidrug‐resistant and multidrug‐sensitive leukemia cells were quantified, and the results showed that o‐SWNTs could be used as the potential drug carriers to deliver small molecules into cells via the physical adsorption along with the circumventing of multidrug resistance of leukemia cells.     

AMPA, not NMDA, activates RhoA GTPases and subsequently phosphorylates moesin

Glutamate induced rapid phosphorylation of moesin, one of ERM family proteins involved in the ligation of membrane to actin cytoskeleton, in rat hippocampal cells (JBC, 277:16576-16584, 2002). However, the identity of glutamate receptor has not been explored. Here we show that a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is responsible for glutamate-induced RhoA activation and phosphorylation of moesin. Glutamate induced phosphorylation at Thr-558 of moesin was still detectible upon chelation of Ca(2+), suggesting involvement of AMPA receptor instead of N-methyl D-Aspartate (NMDA) receptor in this phosphorylation of moesin. AMPA but not NMDA- induced moesin phosphorylation was independent of Ca(2+). Both AMPA and NMDA but not Kainate induced moesin phosphorylation at similar levels. However, the kinetics of phosphorylation varied greatly between AMPA and NMDA where AMPA treatment rapidly increased phosphomoesin, which reached a maximum at 10 min after treatment and returned to a basal level at 30 min. In contrast, NMDA-induced phosphorylation of moesin reached a maximum at 30 min after treatment and was remained at higher levels at 60 min. A possible involvement of RhoA and its downstream effector, Rho kinase in the AMPA receptor-triggered phosphorylation of moesin was also explored. The kinetics for the glutamate- induced membrane translocation of RhoA was similar to that of moesin phosphorylation induced by AMPA. Moreover, Y-27632, a specific Rho kinase inhibitor, completely blocked AMPA-induced moesin phosphorylation but had no effect on NMDA-induced moesin phosphorylation. These results suggest that glutamate-induced phosphorylation of moesin may be mediated through the AMPA receptor/RhoA/Rho kinase pathway.     

A Noncovalent Fluorescence Turn‐on Strategy for Hypoxia Imaging

Hypoxia plays crucial roles in many diseases and is a central target for them. Present hypoxia imaging is restricted to the covalent approach, which needs tedious synthesis. In this work, a new supramolecular host–guest approach, based on the complexation of a hypoxia‐responsive macrocycle with a commercial dye, is proposed. To exemplify the strategy, a carboxyl‐modified azocalix[4]arene (CAC4A) was designed that binds to rhodamine 123 (Rho123) and quenches its fluorescence. The azo groups of CAC4A were selectively reduced under hypoxia, leading to the release of Rho123 and recovery of its fluorescence. The noncovalent strategy was validated through hypoxia imaging in living cells treated with the CAC4A–Rho123 reporter pair.     

Determination of rhodamine 123 by sequential injection technique for pharmacokinetic studies in the rat placenta

The sequential injection (SIA) technique was applied in pharmacokinetic studies of the transporter-mediated passage of a model substrate, rhodamine 123 (Rho123), through the dually perfused rat term placenta. The method described was used for real-time monitoring of Rho123 concentrations in both the maternal and fetal compartments. Determination was processed by fluorescence detection (λ_ex=480 nm, λ_em580 nm); calibration curve was linear over the range 0.01–50 μmol l⁻¹ (r=0.99965), detection limit was 10 nmol l⁻¹ (3σ) and RSD2% (10 readings). Transport of Rho123 was scanned under various conditions (ATP-synthesis inhibition) and several inhibitors of P-glycoprotein transporter were tested (e.g. quinidine). The advantages of the modern SIA method—an automated analytical tool providing both fast and precise analysis—were successfully demonstrated for examination of transport profiles to investigate the effect of P-glycoprotein on the placental transfer of Rho123.     

IS RHODAMINE 123 A PHOTOSENSITIZER?

Because of conflicting reports on the photoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in several in vitro systems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cyt c degradation by an oxygen-independent process. When delivered to skin fibroblasts where it specifically stains mitochondria, Rh 123 photosensitizes membrane damage. These results are consistent with the hypothesis that Rh 123 is a phototoxic stain. The lack of photosensitivity of Rh 123-stained mitochondria in some cell lines might therefore be due to specific structural features.     

A Bifunctional Photosensitizer for Enhanced Fractional Photodynamic Therapy: Singlet Oxygen Generation in the Presence and Absence of Light

The photosensitized generation of singlet oxygen within tumor tissues during photodynamic therapy (PDT) is self‐limiting, as the already low oxygen concentrations within tumors is further diminished during the process. In certain applications, to minimize photoinduced hypoxia the light is introduced intermittently (fractional PDT) to allow time for the replenishment of cellular oxygen. This condition extends the time required for effective therapy. Herein, we demonstrated that a photosensitizer with an additional 2‐pyridone module for trapping singlet oxygen would be useful in fractional PDT. Thus, in the light cycle, the endoperoxide of 2‐pyridone is generated along with singlet oxygen. In the dark cycle, the endoperoxide undergoes thermal cycloreversion to produce singlet oxygen, regenerating the 2‐pyridone module. As a result, the photodynamic process can continue in the dark as well as in the light cycles. Cell‐culture studies validated this working principle in vitro.     

Uncommon behavior of the p-GaAs electrode during the reduction of oxygen in liquid acidic ammonia

In liquid acidic ammonia, the mechanism of oxygen reduction begins as in acidic aqueous solutions, by the successive contribution of the conduction band and then of the valence band (doubling effect). However, unlike in the acidic aqueous solutions, soon after the oxygen photoreduction, an activation process of the p-GaAs surface is initiated, tied to the presence of protons in the solution. This activation of the surface allows the passage from a reduction mechanism via a conduction band to a reduction mechanism via a valence band (oxygen reduction in the dark).     

Competitive immunoassay for Ochratoxin a based on FRET from quantum dot-labeled antibody to rhodamine-coated magnetic silica nanoparticles

We report on a highly sensitive competitive immunoassay for the mycotoxin Ochratoxin (OTA) using magnetic silica nanoparticles (NPs) fluorescently labeled with rhodamine 123 (Rho123) as signal intensifier. The method is based on the measurement of fluorescence resonance energy transfer (FRET) that occurs from CdTe quantum dots covered with anti-OTA antibody to the dye Rho123 on the surface of the NPs. The immunoreaction between anti-OTA antibody and OTA brings the fluorophore (acting as the acceptor) in close proximity of the QDs (acting as the donor), and this causes FRET to occur upon photo-excitation of the QDs. The size and polydispersity of the silica coated magnetic NPs was studied via TEM. The method has a detection limit of 0.8 pg of OTA per mL. It was applied to the determination of OTA in spiked human serum. A linear relationship is found between the increase in the fluorescence intensity of Rho 123 at 580 nm and the concentration of OTA in spiked samples over the 8 to 48 pg?mL^?1 concentration range. This highly sensitive homogeneous competitive detection scheme is simple, rapid and efficient. It does not require multiple separation steps and excessive washing.

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Graphical abstract Following photoexcitation of immobilized quantum dots (QDs), FRET occurs between the QDs and Rhodamine 123. The close proximity of Rho 123 and the magnetic silica core/shell particles leads to strongly intensified emission to result in an assay for Ochratoxin A that has a detection limit as low as 0.8 pg?mL^-1

     

EFFECTIVE PHOTODYNAMIC ACTION BY RHODAMINE 123 LEADING TO PHOTOSENSITIZED KILLING OF CHINESE HAMSTER OVARY CELLS IN TISSUE CULTURE AND A PROPOSED MECHANISM

The effectiveness of rhodamine 123 (R123) as a photosensitizer of cell killing is relatively low and correlates with its inefficient production of singlet oxygen. The known selective retention of R123 in the mitochondria of epithelially derived carcinoma cells, however, is a selective feature that could lead to a more useful therapeutic ratio if photosensitizing effectiveness could be increased. Chinese hamster ovary (CHO) cells in tissue culture were therefore exposed to R123 shortly before and during illumination under conditions controlled for oxygen concentration and temperature. Effective photosensitization of cell killing, as judged by colony formation, was produced by 95% but not by 19% O₂ during illumination of cells at 5d?C or 37d?C, and this was additionally enhanced at the sublethal temperature of 42d?C. Two CHO cell lines were examined; one line, CHO-AA8, was proficient in the repair of DNA damage and the parent to the second line, CHO-EM9, that was deficient in the repair of DNA strand breaks. Cells of both lines incorporated R123 to a similar degree and were similarly photosensitized by the presence of igh oxygen concentration. Furthermore, plasma membrane damage as judged by teh exclusion of trypan blue was not observed immediately after illumination in the presence of R123, but was seen in the presence of meso-tetra-(4-sulfonatophenyl)-porphine (TPPS₄). The extent of damage to the plasma membrane by TPPS₄ was greater in the presence of 95% compared to 19% O₂ during illumination. Photodynamic action at the level of teh plasma membrane appears to contribute to photosensitization by TPPS₄ but not by R123 soon after exposure of cells to these sensitizers. It is hypothesized that photodynamic action by R123 is the primary mechanism causing the observed photosensitization of cell killing, and that mitochondria are teh site of photosensitized damage responsible for this killing.     

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.     

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

Identification of a Single Phosphorylation Site Within Octopus Rhodopsin

Abstract— Light-dependent phosphorylation of rhodopsin (Rho) is a first step in the desensitization of the signaling state of the receptor during vertebrate and invertebrate visual transduction. We found that only ³⁵⁸Ser of the photoac-tivated octopus Rho (oRho*) was phosphorylated by octopus rhodopsin kinase (oRK). Tryptic truncation of the C-terminal PPQGY repeats of oRho that follow the phosphorylation region did not influence spectral or G-protein activation properties of oRho but abolished phos phorylation. Despite significant structural differences between oRK and mammalian RK, these results provide i further evidence of the importance of singly phosphorylated species of Rho* in the generation of arrestin binding sites.     

Interactions between photosynthesis and 'light-enhanced dark respiration' (LEDR) in the flagellate Euglena gracilis after irradiation with ultraviolet radiation

The effects of ultraviolet radiation (UV-A, 315-400 nm plus UV-B, 280-315 nm) on photosynthesis and 'light-enhanced dark respiration' (LEDR) in Euglena gracilis have been investigated by using light pulses (80 s) with increasing photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 micromol m(-2) s(-1) and dark periods between the light pulses. LEDR is estimated as the maximum rate of oxygen consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate. Without any exposure to UV radiation, the photosynthetic rate and LEDR increase with increasing photon fluence rate. After 20 and 40 min exposures to UV radiation, the photosynthetic rate and LEDR as functions of photon fluence rate are reduced. After a 20 min UV treatment respiration is greater than photosynthesis after the first light pulse of 59 micromol m(-2) s(-1) radiation, and especially at higher photon fluence rates photosynthesis is lower than the control values. The inhibitory effects of UV radiation on photosynthetic rate and LEDR are greater after a 40 min UV exposure than after a 20 min exposure. Only at 600 micromol m(-2) s(-1) is the rate of oxygen evolution greater than that of oxygen consumption after a 40 min UV treatment. Both photosynthetic rate and LEDR are inhibited by the photosynthetic inhibitor DCMU (10(-5) M) in a similar way, which indicates close regulatory interactions between photosynthesis and LEDR. Potassium cyanide (KCN) inhibits dark respiration more than it inhibits LEDR. Dark respiration is not affected to the same degree by UV radiation as are photosynthesis and LEDR.     

PHOTOSENSITIVE PHENOMENA OF NITRILE HYDRATASE OF Rhodococcus sp. N-771

Abstract— When the washed cells of Rhodococcus sp. N-771 were incubated at 5°C in the dark under aerobic condition, their nitrile hydratase was inactivated after several days. Most of this activity was recovered by light irradiation. The speed of inactivation in the dark was affected by incubation temperature and amount of oxygen supply. Under anerobic conditions, however, this reversible dark inactivation was not observed; the photoirradiation of the cells irreversibly inactivated the initial cell activity by about 15%. The enzyme activity of the cell-free extract of the inactivated cells can also be recovered when photoirradiated. This process did not require oxygen, and was not prevented by dialysis. However, the enzyme of the cell-free extract could not be inactivated by dark, aerobic incubation nor by photoirradiation after dark anaerobic incubation.     

Effect of desaspidin and DCMU on photokinesis of blue-green algae

Abstract— The effect of desaspidin and DCMU (3–(3.4-dichlorophenyl)-1,1-dimethylurea) on the speed of movement in light and dark of the blue-green alga Phormidium uncinatum has been investigated. Desaspidin, which uncouples oxidative phosphorylation and predominantly cyclic photosynthetic phosphorylations, inhibits movement in the dark and light as well, but dark movement is more sensitive. Movement in the light is more sensitive under anaerobic conditions than in air. The inhibitory effect of desaspidin is markedly increased by DCMU, which inhibits non-cyclic electron transport and coupled phosphorylation in air as well as under argon. There is no evidence for any photodestruction of desaspidin in air, provided that no ferricyanide is present in the medium. These findings are interpreted to confirm the concept that photokinesis (i.e. a light induced acceleration of movement in microorganisms) is the result of an increased ATP production by photosynthetic phosphorylation and that both cyclic and non-cyclic photophosphorylations supply energy for the movement in blue-green algae.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing α-smooth muscle actin (α-SMA) via transforming growth factor-β1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced α-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced α-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced α-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-β1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-β1/Smad2 signaling pathway.     

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H₂O₂ for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.     

P123-PMMA dual-templating generation and unique physicochemical properties of three-dimensionally ordered macroporous iron oxides with nanovoids in the crystalline walls

Three-dimensionally (3D) ordered macroporous (3DOM) iron oxides with nanovoids in the rhombohedrally crystallized macroporous walls were fabricated by adopting the dual-templating [Pluronic P123 and poly(methyl methacrylate) (PMMA) colloidal microspheres] strategy with ferric nitrate as the metal precursor in an ethanol or ethylene glycol and methanol mixed solution and after calcination at 550 °C. The possible formation mechanisms of such architectured materials were discussed. The physicochemical properties of the materials were characterized by means of techniques such as XRD, TGA/DSC, FT-IR, BET, HRSEM, HRTEM/SAED, UV-vis, XPS, and H(2)-TPR. The catalytic properties of the materials were also examined using toluene oxidation as a probe reaction. It is shown that 3DOM-structured α-Fe(2)O(3) without nanovoids in the macroporous walls was formed in the absence of P123 during the fabrication process, whereas the dual-templating strategy gave rise to α-Fe(2)O(3) materials that possessed high-quality 3DOM structures with the presence of nanovoids in the polycrystalline macropore walls and higher surface areas (32-46 m(2)/g). The surfactant P123 played a key role in the generation of nanovoids within the walls of the 3DOM-architectured iron oxides. There was the presence of multivalent iron ions and adsorbed oxygen species on the surface of each sample, with the trivalent iron ion and oxygen adspecies concentrations being different from sample to sample. The dual-templating fabricated iron oxide samples exhibited much better low-temperature reducibility than the bulk counterpart. The copresence of a 3DOM-structured skeleton and nanovoids in the macropore walls gave rise to a drop in the band-gap energy of iron oxide. The higher oxygen adspecies amounts, larger surface areas, better low-temperature reducibility, and unique nanovoid-containing 3DOM structures of the iron oxide materials accounted for their excellent catalytic performance in the oxidation of toluene.