Mitochondrial dysfunction mediates neuronal cell response to DMMB photodynamic therapy.

Photodynamic therapy (PDT) is a process in which a photosensitizer (PS) is exposed to specific wavelengths and generates reactive oxygen species (ROS) which act within nanometers. The low invasive nature and directed cytotoxicity of this approach render it attractive to the treatment of different conditions, including the ones that affect the central nervous system (CNS). The effect of PDT on healthy neurons is one main concern over its use in the CNS, since neuronal-like cells were shown to be particularly sensitive to certain PSs. Among available PSs, 1,9-dimethyl-methylene blue (DMMB) stands out as being resistant to reduction to its inactive leuco form and by being able to produce high levels of singlet­oxygen. In this study, we aimed to investigate DMMB photodamage mechanisms in the hippocampal cell line HT22. Our results demonstrate that DMMB-PDT decrease in cell viability was linked with an increase in cell death and overall ROS production. Besides, it resulted in a significant increase in mitochondrial ROS production and decreased mitochondria membrane potential. Furthermore, DMMB-PDT significantly increased the presence of acidic autolysosomes, which was accompanied by an increase in ATG1 and ATG8 homologue GaBarap1 expression, and decreased DRAM1 expression. Taken together our results indicated that mitochondrial and autophagic dysfunction underlie DMMB-PDT cytotoxicity in neuronal cells.

Photosensitizing properties and subcellular localisation of 3,4-dihydro-ß-carbolines harmaline and harmalol.

Harmaline (1) and harmalol (2) represent two 3,4-dihydro-ß-carboline (DHßCs) most frequently reported in a vast number of living systems. Fundamental aspects including the photosensitizing properties, cellular uptake, as well as the cyto- and phototoxicity of 1 and 2 were investigated herein. The molecular basis underlying the investigated processes are elucidated. Data reveal that both alkaloids show a distinctive pattern of extracellular DNA photodamage. Compound 1 induces a DNA photodamage profile dominated by oxidised purines and sites of base loss (AP sites), whereas 2 mostly induces single-strand breaks (SSBs) in addition to a small extent of purine oxidative damage. In both cases, DNA oxidative damage would occur through type I mechanism. In addition, a concerted hydrolytic attack is suggested as an extra mechanism accounting for the SSBs formation photoinduced by 2. Subcellular internalisation, cyto- and phototoxicity of 1 and 2 and the corresponding full-aromatic derivatives harmine (3) and harmol (4) also showed quite distinctive patterns in a structure-dependent manner. These results are discussed in the framework of the potential biological, biomedical and/or pharmacological roles reported for these alkaloids. The subtle structural difference (i.e., the exchange of a methoxy group for a hydroxyl substituent at C(7)) between harmaline and harmalol, gives rise to distinctive photosensitizing and subcellular localisation patterns.

Combination of Curcumin and Photodynamic Therapy Based on the Use of Red Light or Near-Infrared Radiation in Cancer: A Systematic Review.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic intervention that can be applied to cancer treatment. The interaction between a photosensitizer (PS), ideal wavelength radiation, and tissue molecular oxygen triggers a series of photochemical reactions responsible for producing reactive oxygen species. These highly reactive species can decrease proliferation and induce tumor cell death. The search for PS of natural origin extracted from plants becomes relevant, as they have photoactivation capacity, preferentially targeting tumor cells and because they do not present any or little toxicity to healthy cells. OBJECTIVE: Our work aimed to carry out a qualitative systematic review to investigate the effects of curcumin (CUR), a molecule considered as PS of natural origin, on PDT, using red light or near-infrared radiation in tumor models. METHODS: A systematic search was performed in three databases (PubMed, Scopus, and Web of Science) using the PICOT method, retrieving a total of 1,373 occurrences. At the end of the peer screening, 25 eligible articles were included in this systematic review using inclusion, exclusion, and eligibility criteria. RESULTS: CUR, whether in its free state, associated with metal complexes or other PS and in a nanocarrier system, was considered a relevant PS for PDT using red light or near-infrared against tumoral models in vitro and in vivo, acting by increasing cytotoxicity, inhibiting proliferation, inducing cell death mainly by apoptosis, and changing oxidative parameters. CONCLUSION: The results found in this systematic review suggest the potential use of CUR as a PS of natural origin to be applied in PDT against many neoplasms, encouraging further search in PDT against cancer and serving as an investigative basis for upcoming pre-clinical and clinical applications.

Protoporphyrin IX (PpIX) loaded PLGA nanoparticles for topical Photodynamic Therapy of melanoma cells.

INTRODUCTION: Nanoparticles (Np) can increase drug efficacy and overcome problems associated with solubility and aggregation in a solution of PpIX. PURPOSE: Evaluate if Np interferes in the photophysical and photobiological capacity of the PpIX comparing with free PpIX intended for topical PDT of melanoma. METHODS: In vitro photophysical evaluation of Np-PpIX was carried out through singlet oxygen (1O2) quantum yield. In vitro cytotoxicity and phototoxicity assays have used murine melanoma cell culture. RESULTS: The quantum yield of singlet oxygen has shown that Np did not influence the formation capacity of this reactive species. In the dark, all PpIX-Nps concentrations were less cytotoxic compared to free drugs. At a higher light dose (1500 mJ.cm2) 3.91 µg / mL PpIX had similar % viable cells for free and Np (∼34 %) meaning Nps did not interfere in the photodynamic effect of PpIX. However, at 7.91 µg / mL the phototoxicity increased for both (5.8 % viable cells for free versus 21.7 % for Nps). Despite the higher phototoxicity of free PpIX at this concentration, greater cytotoxicity in the dark was obtained (∼49 % viable cells for free versus ∼90.6 % Np) which means Nps protect the tumor tissue from the photodynamic action of PpIX. CONCLUSIONS: Np is a potential delivery system for melanoma skin cancer, since it maintained the photophysical properties of PpIX and excellent in vitro phototoxicity effect against melanoma cells, reducing cell viability ∼80 % (7.91 µg / mL PpIX in Nps) and provides safe PDT (due to lower cytotoxicity in the dark).

Hypericin in photobiological assays: An overview.

Hypericin is considered a potent photosensitizer for use in antitumor and antimicrobial photodynamic therapy (PDT). This review presents the primary biological results obtained with hypericin in photodynamic therapy applications, such as photodynamic cancer treatment, photoinactivation of microorganisms (PDI), tissue scarring, and photo diagnosis. We present a compilation of in vitro results that have been published thus far; for these studies, we highlight the hypericin concentration, light dose, and other experimental conditions to evaluate the efficiency of photodynamic treatment like cell death, cell viability, or cell proliferation. The results indicate that different hypericin phototoxicity levels can be observed according to the specific light dose and concentration. Furthermore, it was shown that cellular localization and cell death mechanisms (apoptosis and necrosis) are dependent on the cell type.

Bacterial phototoxicity of biomimetic CdTe-GSH quantum dots.

AIM: Fluorescent semiconductor nanoparticles or quantum dots (QDs) have excellent properties as photosensitizers in photodynamic therapy. This is mainly a consequence of their nanometric size and the generation of light-activated redox species. In previous works, we have reported the low-cost biomimetic synthesis of glutathione (GSH) capped QDs (CdTe-GSH QDs) with high biocompatibility. However, no studies have been performed to determine their phototoxic effect. The aim of this work was to characterize the light-induced toxicity of green (QDs500 ) and red (QDs600 ) QDs in Escherichia coli, and to study the molecular mechanism involved. METHODS AND RESULTS: Photodegradation and reduction power of biomimetic QDs was determined to analyse their potential for radical generation. Escherichia coli cells were exposed to photoactivated QDs and viability was evaluated at different times. High toxicity was determined in E. coli cells exposed to photoactivated QDs, particularly QDs500 . The molecular mechanism involved in QDs phototoxicity was studied by determining Cd2+ -release and intracellular reactive oxygen species (ROS). Cells exposed to photoactivated QDs500 presented high levels of ROS. Cells exposed to photoactivated QDs500 presented high levels of ROS. Finally, to understand this phenomenon and the importance of oxidative and cadmium-stress in QDs-mediated phototoxicity, experiments were performed in E. coli mutants in ROS and Cd2+ response genes. As expected, E. coli mutants in ROS response genes were more sensitive than the wt strain to photoactivated QDs, with a higher effect in green-QDs500 . No increase in phototoxicity was observed in cadmium-related mutants. CONCLUSION: Obtained results indicate that light exposure increases the toxicity of biomimetic QDs on E. coli cells. The mechanism of bacterial phototoxicity of biomimetic CdTe-GSH QDs is mostly associated with ROS generation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented establish biomimetic CdTe-GSH QDs as a promising cost-effective alternative against microbial infections, particularly QDs500 .

Uptake of microalgae as sublethal biomarker reveals phototoxicity of oxytetracycline to the crustacean Daphnia magna.

Antibiotics are considered emerging pollutants as their presence in the environment is increasingly common. Although their environmental concentrations are generally low, they can pose risk to organisms through bioaccumulation, causing sublethal effects. Furthermore, solar radiation can trigger reactions in certain compounds after their accumulation within organisms or in the environment. Toxicity and photoinduced toxicity of oxytetracycline (OTC, widely used antibiotic in salmon aquaculture) on Daphnia magna (Crustacea, Cladocera) and microalgae Raphidocelis subcapitata (Chlorophyceae) as its food source was assessed via aqueous exposure. Also, the impact via diet (microalga) to the crustacean was examined. In addition to lethal (immobility) effect, in vivo chlorophyll fluorescence techniques were used to determine food ingestion (gut content as a biomarker of physiological health) in D. magna and physiological status of microalgae. OTC (≤10 mg L - 1) was not acutely (24 h) toxic to R. subcapitata when measured as maximum quantum yield (Fv/Fm) in darkness. However, under short (1 h) UV exposure OTC caused irreversible decrease of Fv/Fm (50%) at ≥0.5 mg L - 1. OTC was not acutely lethal to D. magna (≤10 mg L - 1), however, sublethal effects (43% decrease in food ingestion) at 10 mg L - 1 were demonstrated. UV exposure (4.5 h) strongly exacerbated toxicity of OTC, leading to lethal (87% immobility) and sublethal (81% decrease of feeding in survived individuals) effects. Uptake of OTC (aqueous exposure) and its photosensitization in tissues of D. magna under UV exposure was confirmed. On the other hand, rapid bioadsorption of OTC on cell surface was evident in R. subcapitata. Uptake of OTC in D. magna through diet could not be confirmed at short-term. Photomodification of OTC under UV exposure was observed through changes in its absorption spectrum. The results show that short exposure to summer UV levels of southern Chile can rapidly induce phototoxicity of OTC, suggesting a potential risk to aquatic organisms.

Co-encapsulation of sodium diethyldithiocarbamate (DETC) and zinc phthalocyanine (ZnPc) in liposomes promotes increases phototoxic activity against (MDA-MB 231) human breast cancer cells.

There has been considerable interest in the development of novel photosensitisers for photodynamic therapy (PDT). The use of liposomes as drug delivery systems containing simultaneously two or more drugs is an attractive idea to create a new platform for PDT application. Therefore, the aim of this study was to evaluate the synergistic effect of diethyldithiocarbamate (DETC) and zinc phthalocyanine (PDT) co-encapsulated in liposomes. The reverse-phase evaporation method resulted in the successful encapsulation of DETC and ZnPc in liposomes, with encapsulation efficiencies above 85 %, mean size of 308 nm, and zeta potential of - 36 mV. The co-encapsulation decreased the cytotoxic effects in mouse embryo fibroblast (NIH3T3) cells and inhibited damage to human erythrocytes compared to free DETC + ZnPc. In addition, both the free drugs and co-encapsulated ones promoted more pronounced phototoxic effects on human breast cancer cells (MDA-MB231) compared to treatment with ZnPc alone. This synergistic effect was determined by DETC-induced decreases in the antioxidant enzyme activity of superoxide dismutase (SOD) and glutathione (GSH).

Insights and controversies on sunscreen safety.

Although sunlight provides several benefits, ultraviolet (UV) radiation plays an important role in the development of various skin damages such as erythema, photoaging, and photocarcinogenesis. Despite cells having endogenous defense systems, damaged DNA may not be efficiently repaired at chronic exposure. In this sense, it is necessary to use artificial defense strategies such as sunscreen formulations. UV filters should scatter, reflect, or absorb solar UV radiation in order to prevent direct or indirect DNA lesions. However, the safety of UV filters is a matter of concern due to several controversies reported in literature, such as endocrine alterations, allergies, increased oxidative stress, phototoxic events, among others. Despite these controversies, the way in which sunscreens are tested is essential to ensure safety. Sunscreen regulation includes mandatory test for phototoxicity, but photogenotoxicity testing is not recommended as a part of the standard photosafety testing program. Although available photobiological tests are still the first approach to assess photosafety, they are limited. Some existing tests do not always provide reliable results, mainly due to limitations regarding the nature of the assessed phototoxic effect, cell UV sensitivity, and the irradiation protocols. These aspects bring queries regarding the safety of sunscreen wide use and suggest the demand for the development of robust and efficient in vitro screening tests to overcome the existing limitations. In this way, Saccharomyces cerevisiae has stood out as a promising model to fill the gaps in photobiology and to complete the mandatory tests enabling a more extensive and robust photosafety assessment.

Fucoxanthin for Topical Administration, a Phototoxic vs. Photoprotective Potential in a Tiered Strategy Assessed by In Vitro Methods.

Fucoxanthin possesses a well-described antioxidant activity that might be useful for human skin photoprotection. However, there is a lack of scientific information regarding its properties when applied onto human skin. Thus, the objective of the present study was to assess the photoprotective and phototoxicity potential of fucoxanthin based on its ultraviolet (UVB 280-320 nm; UVA 320-400 nm) and visible (VIS 400-700 nm) absorption, photostability, phototoxicity in 3T3 mouse fibroblast culture vs. full-thickness reconstructed human skin (RHS), and its ability to inhibit reactive oxygen species formation that is induced by UVA on HaCaT keratinocytes. Later, we evaluated the antioxidant properties of the sunscreen formulation plus 0.5% fucoxanthin onto RHS to confirm its bioavailability and antioxidant potential through the skin layers. The compound was isolated from the alga Desmarestia anceps. Fucoxanthin, despite presenting chemical photo-instability (dose 6 J/cm2: 35% UVA and 21% VIS absorbance reduction), showed acceptable photodegradation (dose 27.5 J/cm2: 5.8% UVB and 12.5% UVA absorbance reduction) when it was added to a sunscreen at 0.5% (w/v). In addition, it increased by 72% of the total sunscreen UV absorption spectra, presenting UV-booster properties. Fucoxanthin presented phototoxic potential in 3T3 fibroblasts (mean photo effect 0.917), but it was non-phototoxic in the RHS model due to barrier function that was provided by the stratum corneum. In addition, it showed a significant inhibition of ROS formation at 0.01% (p < 0.001), in HaCat, and in a sunscreen at 0.5% (w/v) (p < 0.001), in RHS. In conclusion, in vitro results showed fucoxanthin protective potential to the skin that might contribute to improving the photoprotective potential of sunscreens in vivo.

Evaluating Hemolytic and Photo Hemolytic Potential of Organophosphorus by In Vitro Method as an Alternative Tool Using Human Erythrocytes.

AIMS: The present study aims to determine the phototoxic and haemolytic activity of organophosphorus. The use of alternative in vitro assays with human erythrocytes is suggested to predict the polluting effect of these products on health. METHODOLOGY: Human erythrocytes from Toluca Blood Bank were used. Sodium dodecyl sulfate was employed as a positive control. Additionally, the haemolysis percentage of three organophosphate (Acetate, Chlorpyrifos, Malathion, Methamidophos, Methyl Parathion) induced photo haemolysis formulated with surfactants on a concentration of 2 x 109 erythrocytes were evaluated. Finally, the products were classified as irritant or phototoxic. RESULTS: Results showed that the HC50 red blood cells were similar for each organophosphate (Malathion and Methamidophos) indicating very irritant action with ratio classification (L/D) of 0.041 and 0.053, respectively. On the other hand, Chlorpyrifos was classified as an irritant with L/D= 0.14. On the other hand, the HC50 obtained photo hemolysis assays irradiated red blood cells was similar for each organophosphate (Acetate, Chlorpyrifos, Malathion, Methamidophos, Methyl Parathion) indicating no phototoxic action. CONCLUSION: As a conclusion, it can be said that the parameters of haemolysis and denaturation of proteins are good indicators to classify organophosphorus formulated with surfactants as irritating or phototoxic.

Maculopatia solar após o uso de LSD: relato de caso / Solar maculopathy after LSD use: a case report

Introdução: A maculopatia ou retinopatia solar é uma lesão foto-traumática da mácula causada pela observação direta ou indireta de fontes luminosas intensas, que ocorre comumente na presença de distúrbios psíquicos ou após o uso de drogas recreativas. O prognóstico visual varia e a conduta é expectante. Descrição do caso: Paciente V.V.A.M., sexo masculino, 20 anos, estudante, com queixa de escotoma central em ambos os olhos. Nega antecedentes patológicos e oculares. Solicitaram-se tomografia de coerência óptica (OCT) e retinografia, que revelaram uma lesão central, bilateral e simétrica na retina externa. Paciente relatou ter feito uso de Dietilamida de ácido lisérgico (LSD) e, sob influência da droga, ter olhado de forma direta para o sol por aproximadamente 40 minutos. Discussão: O prognóstico da retinopatia solar é variável e relaciona-se com o tempo de exposição e com o comprimento da onda da fonte de luz. A etiopatogênese é explicada pelo dano causado ao epitélio pigmentar da retina (EPR) pela radiação. Conclusões: Deve haver maior orientação ao público sobre os possíveis efeitos danosos de exposição a fontes de luz de origens diversas. Além disso, destaca-se a importância do OCT para a identificação da maculopatia solar. (AU)

Introduction: Solar maculopathy or retinopathy is photo-traumatic damage created on the macula, caused by direct or indirect observation of intense light sources, commonly occurring in the presence of psychic disorders or after the use of recreational drugs. The visual prognosis varies. There is currently no known treatment. Case report: A 20-year-old male with no previous complaints reported central scotoma in both eyes despite 20/20 uncorrected vision. Bilateral, symmetric, central changes could be seen in the macula in fundoscopy. Optical coherence tomography (OCT) confirmed loss of the external retina suggestive of Solar Maculopathy. The patient later claimed to have spent 40 minutes looking directly into the sun after use of Lysergic Acid Diethylamide (LSD). Discussion: The prognosis of solar retinopathy is related to the exposure time and to the wavelength of the light source, with those between 300-350 nm being the most harmful. Its etiopathogenesis is explained by damage caused to the retinal pigment epithelium (EPR) caused by radiation, interrupting the interdigitations between this layer and the external segment of the photoreceptors. Ophthalmoscopically, solar maculopathy is characterized by a small foveolar lesion that might become yellowish in the days following exposure, in the form of exudate or edema, followed by loss of foveal reflex and thinning of the fovea. The initial yellowed lesions are subsequently replaced by a spotted EPR or even by a lamellar orifice. Conclusions: There should be public guidance on the possible harmful effects of exposure to sources of light from diverse origins, as it usually occurs during solar eclipses, after exposure to certain types of lasers or observation of fires since this habit can cause severe and sometimes irreversible visual loss. (AU)

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

Spirulina, Palmaria Palmata, Cichorium Intybus, and Medicago Sativa extracts in cosmetic formulations: an integrated approach of in vitro toxicity and in vivo acceptability studies.

Background/Aims: The selection of suitable raw materials in the cosmetic research and development is a key point, not only in order to obtain the expected results but also to avoid undesirable side effects. This study evaluated the in vitro toxicity potential of four different plant extracts and their in vivo acceptability studies. Methods: Spirulina, Palmaria palmata, Cichorium intybus and Medicago sativa extracts were analysed alone or in combination and added in cosmetic formulations. The in vitro toxicity evaluation, Hen's Egg Chorioallantoic Membrane Test (HET-CAM) and 3T3 NRU phototoxicity test were performed to evaluate in vitro potential ocular irritation and photo safety, respectively. Twenty subjects were enrolled in the acceptability studies, who were evaluated for the absence of harmful effects of the formulation by visual assessment and by transepidermal water loss, a biophysical technique, for 30 days. Results: HET-CAM assay showed that the studied extracts added to a gel-cream formulation had no irritant potential. In addition, the combination of Palmaria palmata, alfalfa and chicory extracts did not show phototoxic potential in vitro. Acceptability studies showed that the formulation containing the four extracts combined did not provoke any transepidermal water loss (TEWL) alteration, sensory irritation or erythema in the forearms for the period of analysis. Conclusion: The studied active ingredients, alone or in combination, present no cytotoxicity potential and when added to a gel-cream formulation had no irritant potential in vitro. These results predicting no harmful effects were confirmed in the acceptability tests, which showed no alteration on skin barrier function and no report of irritation perception of sign of erythema, suggesting the potential of these extracts for the development of safe cosmetic products.

Toxicity of blue led light and A2E is associated to mitochondrial dynamics impairment in ARPE-19 cells: implications for age-related macular degeneration.

Age-related macular degeneration (AMD) is a multifactorial retinal disease characterized by a progressive loss of central vision. Retinal pigment epithelium (RPE) degeneration is a critical event in AMD. It has been associated to A2E accumulation, which sensitizes RPE to blue light photodamage. Mitochondrial quality control mechanisms have evolved to ensure mitochondrial integrity and preserve cellular homeostasis. Particularly, mitochondrial dynamics involve the regulation of mitochondrial fission and fusion to preserve a healthy mitochondrial network. The present study aims to clarify the cellular and molecular mechanisms underlying photodamage-induced RPE cell death with particular focus on the involvement of defective mitochondrial dynamics. Light-emitting diodes irradiation (445 ± 18 nm; 4.43 mW/cm2) significantly reduced the viability of both unloaded and A2E-loaded human ARPE-19 cells and increased reactive oxygen species production. A2E along with blue light, triggered apoptosis measured by MC540/PI-flow cytometry and activated caspase-3. Blue light induced mitochondrial fusion/fission imbalance towards mitochondrial fragmentation in both non-loaded and A2E-loaded cells which correlated with the deregulation of mitochondria-shaping proteins level (OPA1, DRP1 and OMA1). To our knowledge, this is the first work reporting that photodamage causes mitochondrial dynamics deregulation in RPE cells. This process could possibly contribute to AMD pathology. Our findings suggest that the regulation of mitochondrial dynamics may be a valuable strategy for treating retinal degeneration diseases, such as AMD.

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. AIMS OF THE STUDY: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. MATERIALS AND METHODS: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. RESULTS: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. CONCLUSION: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

Diagnostic Delay in Erythropoietic Protoporphyria.

Erythropoietic protoporphyria is a photodermatosis presenting in childhood with severe pain on sun exposure. The diagnosis is often delayed because of the lack of awareness among pediatricians. We describe the diagnostic odyssey of 2 children presenting with symptoms of erythropoietic protoporphyria and report results of a survey of 129 affected individuals.

UVA and UVB formulation phototoxicity in a three-dimensional human skin model: Photodegradation effect.

In vitro three-dimensional human skin models are an innovative alternative to evaluate cytotoxicity and phototoxicity in the cosmetic industry. The aim of this study was to use a skin model to evaluate the potential toxicity of sunscreen formulations with or without exposure to UV radiation. In addition, the toxicity of these formulations was evaluated after exposure to photodegradation. The results showed toxicity with all formulations/conditions tested, including the control formulation, compared to PBS. Cell viability of photodegraded formulations - prior to the phototoxicity radiation process - was higher, indicating that some formulation components were degraded into products with reduced toxicity. The results also indicated that avobenzone was more unstable/toxic than octyl p-methoxycinnamate under the same test conditions. The sunscreens and their formulations were shown to be toxic to skin model cells to some extent, even when not exposed to UV irradiation; however the biological role of this toxicity is unclear. This result shows the importance of testing sunscreen formulations in real in-use conditions. Finally, since we used an in vitro assay based on a human cell model, this non-invasive technique represents a suitable alternative to animal models for phototoxicity tests in general and could have application in screening new sunscreen products.

Comparative study of phototoxicity of protoporphyrin IX synthetic and extracted from ssp Rattus novergicus albinus rats toward murine melanoma cells.

Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.