Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Lipofuscin in keratinocytes: Production, properties, and consequences of the photosensitization with visible light.

A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by the maximization of lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (~450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ~1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (1O2) when irradiated with blue light (420 nm), but there was no 1O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.

Effects of methylene blue-mediated photodynamic therapy on a mouse model of squamous cell carcinoma and normal skin.

BACKGROUND: Photodynamic therapy is used to treat a variety of cancers and skin diseases by inducing apoptosis, necrosis, immune system activation, and/or vascular damage. Here, we describe the effects of a single photodynamic therapy session using methylene blue on a mouse model of squamous cell carcinoma and normal skin. METHODS: The photodynamic therapy protocol comprised application of a 1% methylene blue solution, followed by irradiation with a diode laser for 15 min at 74 mW/cm2, for a total dose of 100 J/cm2. Morphological changes, cell proliferation, apoptosis, collagen quantity, immune system activity, and blood vessel number were analyzed 24 h and 15 days after photodynamic therapy. RESULTS: In the squamous cell carcinoma group, photodynamic therapy reduced tumor size and cell proliferation and raised cytokine levels. In normal skin, it decreased cell proliferation and collagen quantity and increased apoptosis and blood vessel numbers. CONCLUSIONS: The effects of photodynamic therapy were greater on normal skin than squamous cell carcinoma tissues. The reduced epithelial thickness and keratinization of the former are factors that contribute to the efficacy of this treatment. Adjustments to the treatment protocol are necessary to potentiate the effects for squamous cell carcinoma therapy.

Direct participation of DNA in the formation of singlet oxygen and base damage under UVA irradiation.

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.

Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage.

The premature aging (photoaging) of skin characterized by wrinkles, a leathery texture and mottled pigmentation is a well-documented consequence of exposure to sunlight. UVA is an important risk factor for human cancer also associated with induction of inflammation, immunosuppression, photoaging and melanogenesis. Although herbal compounds are commonly used as photoprotectants against the harmful effects of UVA, the mechanisms involved in the photodamage are not precisely known. In this study, we investigated the effects of Aloe Vera (Aloe barbadensis mil) on the protection against UVA-modulated cell killing of HaCaT keratinocytes. Aloe Vera exhibited the remarkable ability of reducing both in vitro and in vivo photodamage, even though it does not have anti-radical properties. Interestingly, the protection conferred by Aloe Vera was associated with the maintenance of membrane integrity in both mimetic membranes and intracellular organelles. The increased lysosomal stability led to a decrease in lipofuscinogenesis and cell death. This study explains why Aloe Vera extracts offer protection against photodamage at a cellular level in both the UV and visible spectra, leading to its beneficial use as a supplement in protective dermatological formulations.

Photochemical degradation increases polycyclic aromatic hydrocarbon (PAH) toxicity to the grouper Epinephelus marginatus as assessed by multiple biomarkers.

The effects of halogen-light-irradiated and non-irradiated PAHs on the grouper Epinephelus marginatus were assessed through biomarkers including morphometric parameters, liver histopathology, biliary PAH concentration, genetic alterations, and enzyme activity modulation. E. marginatus juveniles were divided into three groups: control (C), non-irradiated PAHs (PAHs1), and irradiated PAHs (PAHs2). Test groups were exposed for 14 days to a 0.5 ppm PAH solution in the semi-static system. After this period, fish were anesthetized with benzocaine (2%) and peripheric blood was collected by caudal puncture. Blood smears were prepared and stained with propidium iodide. Fish livers were collected, fixed in McDowell's solution, embedded in paraplast, thin-sectioned, and stained with hematoxylin-eosin (H&E). For biochemical analyses including superoxide dismutase, catalase, and glutathione S-transferase activities, fish livers were collected and preserved in liquid nitrogen. Water samples were analyzed using gas chromatography-mass spectrometry (GC-MS) and bile synchronous fluorescence spectroscopy. Fish in the PAHs2 group had micronuclei (MN) in blood cells, as well as significant differences in nuclear morphology (NMA). Significant morphological alterations were observed in the livers from fish exposed to PAHs as well as inhibition of the catalase activity. Our results show that irradiation altered the bioavailability of PAHs, especially benzanthracene, which has great impact in aquatic ecosystems. Among the consequences of physical and chemical changes to PAHs, we observed a significant increase in NMA and MN incidence in E. marginatus erythrocytes, indicating the potential initiation of mutagenic and carcinogenic processes.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔµH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

Melanin photosensitization and the effect of visible light on epithelial cells.

Protecting human skin from sun exposure is a complex issue that involves unclear aspects of the interaction between light and tissue. A persistent misconception is that visible light is safe for the skin, although several lines of evidence suggest otherwise. Here, we show that visible light can damage melanocytes through melanin photosensitization and singlet oxygen (1O2) generation, thus decreasing cell viability, increasing membrane permeability, and causing both DNA photo-oxidation and necro-apoptotic cell death. UVA (355 nm) and visible (532 nm) light photosensitize 1O2 with similar yields, and pheomelanin is more efficient than eumelanin at generating 1O2 and resisting photobleaching. Although melanin can protect against the cellular damage induced by UVB, exposure to visible light leads to pre-mutagenic DNA lesions (i.e., Fpg- and Endo III-sensitive modifications); these DNA lesions may be mutagenic and may cause photoaging, as well as other health problems, such as skin cancer.

Rapid screening of potential autophagic inductor agents using mammalian cell lines.

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

Diguanoside tetraphosphate (Gp4G) is an epithelial cell and hair growth regulator.

Our goal was to study the effect of Gp4G on skin tissues and unravel its intracellular action mechanisms. The effects of Gp4G formulation, a liposomic solution of Artemia salina extract, on several epidermal, depmal, and hair follicle structures were quantified. A 50% increase in hair length and a 30% increase in the number of papilla cells were explained by the changes in the telogen/anagen hair follicle phases. Increasing skin blood vessels and fibroblast activation modified collagen arrangement in dermal tissues. Imunohistochemical staining revealed expressive increases of versican (VER) deposition in the treated animals (68%). Hela and fibroblast cells were used as in vitro models. Gp4G enters both cell lines, with a hyperbolic saturation profile inducing an increase in the viabilities of Hela and fibroblast cells. Intracellular ATP and other nucleotides were quantified in Hela cells showing a 38% increase in intracellular ATP concentration and increases in the intracellular concentration of tri- , di- , and monophosphate nucleosides, changing the usual quasi-equilibrium state of nucleotide concentrations. We propose that this change in nucleotide equilibrium affects several biochemical pathways and explains the cell and tissue activations observed experimentally.

Singlet molecular oxygen trapping by the fluorescent probe diethyl-3,3'-(9,10-anthracenediyl)bisacrylate synthesized by the Heck reaction.

Singlet molecular oxygen O(2)((1)Δ(g)) is a potent oxidant that can react with different biomolecules, including DNA, lipids and proteins. Many polycyclic aromatic hydrocarbons have been studied as O(2)((1)Δ(g)) chemical traps. Nevertheless, a suitable modification in the polycyclic aromatic ring must be made to increase the yield of O(2)((1)Δ(g)) chemical trapping. With this goal, an anthracene derivative, diethyl-3,3'-(9,10-anthracenediyl)bisacrylate (DADB), was obtained from the reaction of 9,10-dibromoanthracene and ethyl acrylate through the Heck coupling reaction. The coupling of ethyl acrylate with the anthracene ring produced a new lipophilic, esterified, fluorescent probe reactive toward O(2)((1)Δ(g)). This compound reacts with O(2)((1)Δ(g)) at a rate of k(r) = 1.69 × 10(6) M(-1) s(-1) forming a stable endoperoxide (DADBO(2)), which was characterized by UV-Vis, fluorescence, HPLC/MS and (1)H and (13)C NMR techniques. The photophysical, photochemical and thermostability features of DADB were also evaluated. Furthermore, this compound has the potential for great application in biological systems because it is easily synthetized in large amount and generates specific endoperoxide (DADBO(2)), which can be easily detected by HPLC tandem mass spectrometry (HPLC/MS/MS).

Generation and suppression of singlet oxygen in hair by photosensitization of melanin.

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (λ(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (λ(em)=1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin.

Langmuir films of petroleum at the air-water interface.

Understanding the behavior of petroleum films at the air/water interface is crucial for dealing with oil slicks and reducing the damages to the environment, which has normally been attempted with studies of Langmuir films made of fractions of petroleum. However, the properties of films from whole petroleum samples may differ considerably from those of individual fractions. Using surface pressure and surface potential measurements and Brewster angle and fluorescence microscopy, we show that petroleum forms a nonhomogeneous Langmuir film at the air-water interface. The surface pressure isotherms for petroleum Langmuir films exhibit gas (G), liquid-expanded (LE), and liquid-condensed phases, with almost no hysteresis in the compression-decompression cycles. Domains formed upon compression from the G to the LE phase were accompanied by an increase in fluorescence intensity with excitation at 400-440 nm owing to an increase in the surface density of the chromophores in the petroleum film. The surface pressure and the fluorescence microscopy data pointed to self-assembling domains into a pseudophase in thermodynamic equilibrium with other less emitting petroleum components. This hypothesis was supported by Brewster angle microscopy images, whereby the appearance of water domains even at high surface pressures confirms the tendency of petroleum to stabilize emulsion systems. The results presented here suggest that, for understanding the interaction with water, it may be more appropriate to use the whole petroleum samples rather than its fractions.

Spectrofluorimetric determination of second critical micellar concentration of SDS and SDS/Brij 30 systems.

Potentially useful stead-state fluorimetric technique was used to determine the critical micellar concentrations (CMC(1) and CMC(2)) for two micellar media, one formed by SDS and the other by SDS/Brij 30. A comparative study based on conductimetric and surfacial tension measurements suggests that the CMC(1) estimated by the fluorimetric method is lower than the value estimated by these other techniques. Equivalent values were observed for SDS micelles without Brij 30 neutral co-surfactant. The use of acridine orange as fluorescent probe permitted to determine both CMC(1) and CMC(2). Based on it an explanation on aspects of micelle formation mechanism is presented, particularly based on a spherical and a rod like structures.

Quenching of singlet molecular oxygen, O2(1Deltag), by dipyridamole and derivatives.

Dipyridamole (DIP) is known for its vasodilating and antiplatelet activity, exhibiting also a potent antioxidant effect, strongly inhibiting lipid peroxidation. This effect has been studied in mitochondria and a correlation between the DIP derivatives' structure, the ability to bind to micelles and biological activity has been suggested. In the present work, the quenching of singlet molecular oxygen, O(2)((1)Delta(g)), by DIP and RA47 and RA25 derivatives was analyzed in acetonitrile (ACN) and aqueous acid solutions. Laser flash photolysis excitation of methylene blue (MB) was made at 532 nm and monomol light emission of O(2)((1)Delta(g)) was monitored at 1270 nm. Bimolecular quenching constants in ACN are consistent with an efficient physical quenching, presenting values a bit lower than the diffusion limit (k(t) = 3.4-6.8 x 10(8) M(-1 )s(-1)). The quenching process probably occurs via reversible charge transfer with the formation of an exciplex. Calculation of DeltaG(et) associated with O(2)((1)Delta(g)) quenching corroborates with uncompleted electron transfer. In aqueous acid solutions (pH = 3.0), the k(t) values for DIP and derivatives are 20-fold smaller when compared with ACN. The electrochemical properties of DIP in ACN are characterized by two consecutive one-electron processes with half-wave oxidation potentials of 0.30 and 0.67 V vs saturated calomel electrode (SCE). However, in an aqueous acid medium, a single oxidation wave is observed involving a two-electron process (0.80 V vs SCE). Therefore, O(2)((1)Delta(g)) quenching is consistent with electrochemical data.

Light-driven horseradish peroxidase cycle by using photo-activated methylene blue as the reducing agent.

In this work, the regeneration of native horseradish peroxidase (HRP), following the consecutive reduction of oxo-ferryl pi-cation (compound I) and oxo-ferryl (compound II) forms, was observed by UV-visible spectrometry and electron paramagnetic resonance (EPR) in the presence of methylene (MB+), in the dark and under irradiation. In the dark, MB+ did not affect the rate of HRP compound I and II reduction, compatible with hydrogen peroxide as the solely reducing species. Under irradiation, the dye promoted a significant increase in the native HRP regeneration rate in a pH-dependent manner. Flash photolysis measurements revealed significant redshift of the MB+ triplet absorbance spectrum in the presence of native HRP. This result is compatible with the dye binding on the enzyme structure leading to the increase in the photogenerated MB* yield. In the presence of HRP compound II, the lifetime of the dye at 520 nm decreased approximately 75% relative to the presence of native HRP that suggests MB* as the heme iron photochemical reducing agent. In argon and in air-saturated media, photoactivated MB+ led to native HRP regeneration in a time- and concentration-dependent manner. The apparent rate constant for photoactivated MB+-promoted native HRP regeneration, in argon and in air-saturated medium and measured as a function of MB+ concentration, exhibited saturation that is suggestive of dye binding on the HRP structure. The dissociation constant (KMB) observed for the binding of dye to HRP was 5.4+/-0.6 microM and 0.57+/-0.05 microM in argon and air-saturated media, respectively. In argon-saturated medium, the rate of the conversion of HRP compound II to native HRP was significantly higher, k2argon=(2.1+/-0.1)x10(-2) s(-1), than that obtained in air-equilibrated medium, k2air=(0.73+/-0.02)x10(-2) s(-1). Under these conditions the efficiency of photoactivated MB(+)-promoted native HRP regeneration was determined in argon and air-equilibrated media as being, respectively: k2/KMB=3.9x10(3) and 12.8x10(3) M(-1) s(-1).

Photo-induced destruction of giant vesicles in methylene blue solutions.

We study the photodecomposition of phospholipid bilayers in aqueous solutions of methylene blue. Observation of giant unilamellar vesicles under an optical microscope reveals a consistent pattern of membrane disruption as a function of methylene blue concentration and photon density for different substrates supporting the vesicles.

Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications.

Methylene blue (MB) is a molecule that has been playing important roles in microbiology and pharmacology for some time. It has been widely used to stain living organisms, to treat methemoglobinemia, and lately it has been considered as a drug for photodynamic therapy (PDT). In this review, we start from the fundamental photophysical, photochemical and photobiological characteristics of this molecule and evolved to show in vitro and in vivo applications related to PDT. The clinical cases shown include treatments of basal cell carcinoma, Kaposi's Sarcoma, melanoma, virus and fungal infections. We concluded that used together with a recently developed continuous light source (RL50(®)), MB has the potential to treat a variety of cancerous and non-cancerous diseases, with low toxicity and no side effects.

Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions.

Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MB's cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.

Solvent effects on the photophysics of 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one.

The photophysics of 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one was studied in different solvents and in SDS micelles. This compound presents characteristics which include an S(0)---> S(1) ( pi,pi*) transition with a (1)(n,pi*) perturbative component, due to the electronic coupling between the diethylamino group and the coumarin ring, considerable solvatochromism, dual fluorescence and high fluorescence quantum yields in almost all solvents studied. The electronic structure of the S(1) and S(2) excited states permits vibronic coupling between them, making configurational changes of the S(2) excited state possible, leading to the formation of an S(2)(TICT) state. Analysis of the TCSPC data indicates an equilibrium between the S(2)(TICT) and S(1)(LE) states in favour of the former. In protic solvents, the hydrogen bonding between the solvent and the diethylamino moiety results in the formation of an S(2)(HICT) state, making internal conversion an important deactivation process. Quantum mechanical calculations for the isolated molecule show that the diethylamino group in the S(2)(TICT) state is twisted at least 56 degree from the plane of the coumarin ring, with partial electronic decoupling between -NEt(2) and the coumarin ring. This twisting angle must be positively influenced by solute-solvent interactions. [capital Phi](ST) is found to be small, but not negligible. However, Phi (delta) can be considered negligible, an indication that T(1) is a short-lived state. Based on the experimental data and theoretical calculations, the most probable sequence for the first excited states, including the TICT state, is T(1)(n, pi*) < S(2)(TICT) < S(1)(pi,pi*) approximate S(2) (n,pi*).