Practical Aspects in the Study of Biological Photosensitization Including Reaction Mechanisms and Product Analyses: A Do's and Don'ts Guide

The interaction of light with natural matter leads to a plethora of photosensitized reactions. These reactions cause the degradation of biomolecules, such as DNA, lipids, proteins, being therefore detrimental to the living organisms, or they can also be beneficial by allowing the treatment of several diseases by photomedicine. Based on the molecular mechanistic understanding of the photosensitization reactions, we propose to classify them in four processes: oxygen-dependent (type I and type II processes) and oxygen-independent [triplet-triplet energy transfer (TTET) and photoadduct formation]. In here, these processes are discussed by considering a wide variety of approaches including time-resolved and steady-state techniques, together with solvent, quencher, and scavenger effects. The main aim of this survey is to provide a description of general techniques and approaches that can be used to investigate photosensitization reactions of biomolecules together with basic recommendations on good practices. Illustration of the suitability of these approaches is provided by the measurement of key biomarkers of singlet oxygen and one-electron oxidation reactions in both isolated and cellular DNA. Our work is an educational review that is mostly addressed to students and beginners.Fil: Baptista, Maurício S.. Universidade de Sao Paulo; BrasilFil: Cadet, Jean. University of Sherbrooke; CanadáFil: Greer, Alexander. City University of New York; Estados UnidosFil: Thomas, Andrés Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin

Photochemistry of Lipofuscin and the Interplay of UVA and Visible Light in Skin Photosensitivity

The topics about prevention against sunlight-induced damages and a secure threshold to light exposition have reached a bigger number of specialists in basic science and medical care. It has been accepted that ultraviolet light is very hazardous and visible light is safe, but recent studies from our group has shown that human keratinocytes exposed previously to ultraviolet A (UVA) light can generate an endogenous visible light-sensitive photosensitizer (lipofuscin), leading to higher levels of singlet oxygen, DNA damages and a wide-range of cellular insults due to intracellular lipofuscin accumulation. Disruption of cell death pathways and on essential metabolic processes, as autophagy and redox signaling, can collaborate to increase light-induced damages. We also discuss the importance of considering not only UVA but visible light too in protection against solar exposure as a way to prevent future pretumoral lesions

Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers

A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized in order to study the role of amphiphilicity and zinc insertion in photodynamic therapy (PDT) efficacy. Several properties of the PS were evaluated and compared within the series including photophysical properties (absorption spectra, fluorescence quantum yield U f , and singlet oxygen quantum yield U D ), uptake by vesicles, mitochondria and HeLa cells, dark and phototoxicity in HeLa cells. The photophysical properties of all compounds are quite similar (U f ≤ 0.02; U D~0 .8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential, i.e., in uncoupled mitochondria PS binding decreases by up to 53%. The porphyrin substituted with octyl groups (TC8PyP) is the compound that is most enriched in mitochondria, and its zinc derivative (ZnTC8PyP) has the highest global uptake. The stronger membrane interaction of the zinc-substituted porphyrins is attributed to a complexing effect with phosphate groups of the phospholipids. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions which allow more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to the membrane binding and cell uptake, but it is not totally related to mitochondrial targeting

Ictiofauna de três praias da baía da Ribeira, Angra dos Reis, Rio de JanEiro

O presente trabalho objetiva descrever a composição, a distribuição, a abundância relativa e a riqueza das espécies de peixes que ocorrem em três praias da Baía da Ribeira, situada no município de Angra dos Reis, Rio de Janeiro. Coletas mensais foram realizadas de dezembro de 1998 a janeiro de 2000 em três áreas: a área 1, localizada na praia da Aroeira, a nordeste da Baía da Ribeira; a área 2, localizada na praia do canal do Bracuí, a oeste; e a área 3, localizada na praia do Saco Piraquara de Fora, a sudoeste. Foram capturados 509 espécimes, compreendendo 17 famílias e 28 espécies. As abundâncias relativas das 6 espécies mais representativas por área de coleta foram: Atherinella brasiliensis com 47,9%, Oligoplites saurus com 12%, Diapterus rhombeus com 9,6%, Gerres aprion com 7,3%, Chirocentrodon bleekerianus com 4,1% e Sphoeroides testudineus com 3,7%. A área 1 apresentou maior abundância relativa com 62,9% do total; a área 2 com 30% e a área 3 com 7,1%. A maior riqueza específica foi observada na área 1 com 6,8, seguida da área 3 com 6,0 e da área 2 com 4,1.   ABSTRACT   Ichthyofauna of three beaches of  Ribeira’s bay, Angra dos Reis, Rio de Janeiro The aim of this paper is to describe the composition, the distribution, the relative abundance and the fishes species wealth that occur in three beaches of Baía da Ribeira, situated in Angra dos Reis’ municipal district, Rio de Janeiro. Monthly collections were accomplished from December 1998 to January 2000 in three areas: the area 1, located in the Aroeira beach, the Baía da Ribeira northeast; the area 2, located in the channel beach of Bracuí, to the west; and the area 3, located in the beach of Saco Piraquara de Fora, the southwest. Five hundred and nine specimens were captured, with 17 families comprehending 28 species. The relative abundance of the six more representative species by the collection areas were: Atherinella brasiliensis with 47.9%, Oligoplites saurus with 12%, Diapterus rhombeus with 9.6%, Eucinostomus aprion with 7.3%, Chirocentrodon bleekerianus with 4.1% and Sphoeroides testudineus with 3.7%. The area 1 showed a larger relative abundance with 62.9% from the total; the area 2 with 30% and the area 3 with 7.1%. Fish-species richness was observed in the area 1 with 6.8, followed by the area 3 with 6.0 and by area 2 with 4.1. Key words: Community, fishes, beaches, bay

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 singlet‑oxygen. 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.</p

Inactivation Kinetics and Lethal Dose Analysis of Antimicrobial Blue Light and Photodynamic Therapy.

BACKGROUND: Photodynamic therapy (PDT) has been long used to treat localized tumors and infections. Currently, microbial inactivation data is reported presenting survival fraction averages and standard errors as discrete points instead of a continuous curve of inactivation kinetics. Standardization of this approach would allow clinical protocols to be introduced globally, instead of the piecemeal situation which currently applies. METHODS: To this end, we used a power-law function to fit inactivation kinetics and directly report values of lethal doses (LD) and a tolerance factor (T) that informs if inactivation rate varies along the irradiation procedure. A deduced formula was also tested to predict LD for any given survival fraction value. We analyzed the photoantimicrobial effect caused by red light activation of methylene blue (MB-APDT) and by blue light (BL) activation of endogenous microbial pigments against 5 clinically relevant pathogens. RESULTS: Following MB- APDT, Escherichia coli and Staphylococcus aureus cells become increasingly more tolerant to inactivation along the irradiation process (T  1). P. aeruginosa and Candida albicans present constant inactivation rate (T˜1). In contrast, all bacterial species presented similar behavior during inactivation caused by BL, i.e., continuously becoming more sensitive to blue light exposure (T > 1). CONCLUSION: The power-law function successfully fit all experimental data. Our proposed method precisely predicted LD and T values. We expect that these analytical models may contribute to more standardized methods for comparisons of photodynamic inactivation efficiencies

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications