Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment / Acumulación celular y efecto anti- Leishmania de la protoporfirina IX exógena y endógena después del tratamiento fotodinámico

Introduction: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. Objective: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. Materials and methods: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. Results were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). Results: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16±0.01mM and 0.33±0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032±0.00002 mM), L. (L.) infantum (IC 50 0.003±0.0001 mM) and L. (V.) panamensis (IC 50 0.024±0.0001 mM) promastigotes. Conclusions: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Introducción. El tratamiento fotodinámico con ácido 5-aminolevulínico como inductor de la protoporfirina IX (ALA-PpIX) constituye una alternativa interesante en el tratamiento de la leishmaniasis cutánea. Objetivo. Evaluar la producción de protoporfirina IX (PpIX) a partir de la administración de ALA o MAL y el efecto de la PDT con ALA a nivel celular en células THP-1 no infectadas e infectadas con Leishmania ( Viannia ) panamensis o Leishmania ( Leishmania ) infantum (syn. Leishmania chagasi ). Materiales y métodos. La producción de protoporfirina IX y su ‘colocalización´ mitocondrial se evaluaron mediante microscopía ‘confocal´. Se evaluó la toxicidad celular después del tratamiento con los compuestos y la aplicación de irradiación de luz (597-752 nm) en una fluencia de 2,5 J/cm 2 mediante el empleo de la prueba colorimétrica con metil-tiazol-tetrazolio (MTT) en las células, y de métodos microscópicos estándar en los parásitos. Los resultados se expresaron como la concentración del compuesto activo en el 50 % de las células o parásitos (CC 50 o CI 50 ). Resultados. El ácido aminolevulínico o el metil-5-aminolevulinato indujeron la protoporfirina IX endógena en células humanas, y se observó fluorescencia de color rojo en las mitocondrias. La actividad del ácido aminolevulínico y del metil-5-aminolevulinato utilizados con terapia fotodinámica fue similar en las células THP-1 (CC 50 0,16±0,01 mM y 0,33±0,019 mM, respectivamente) y, aparentemente, no inhibió los parásitos en las células infectadas, en comparación con los controles. El tratamiento exógeno con protoporfirina IX y terapia fotodinámica fue tóxico para las células THP-1 (CC 50 0,00032 ±0,00002 mM) y para los promastigotes de L. (L .) infantum (IC 50 0,003±0,0001 mM) y L. ( V .) panamensis (CI 50 0,024±0,0001 mM). Conclusiones. A pesar de la ‘fotoactividad´ del tratamiento con protoporfirina IX en promastigotes y de su producción después del tratamiento con ácido aminolevulínico y metil-5-aminolevulinato en las células infectadas con Leishmania , no se observó daño en los amastigotes presentes en las células de L. ( L .) infantum o L . ( V .) panamensis .

Photodynamic inactivation of antibiotic resistant strain of Pseudomonas aeruginosa by porphyrins induced by delta-aminolaevulinic acid.

BACKGROUND & OBJECTIVES: The widespread occurrence of antibiotic resistant strains of Pseudomonas aeruginosa in hospitals is a matter of growing concern. We report the results of a study on photodynamic inactivation of antibiotic resistant strain of P. aeruginosa by delta-aminolaevulinic acid (ALA). METHODS: Exponentially growing P. aeruginosa cells were incubated in growth medium with ALA for various durations. Subsequently, the cells were washed and resuspended in phosphate buffered saline (PBS). These cells were incubated with different concentrations of glutathione (GSH) in PBS for 15 min. Porphyrins synthesized with and without GSH were detected by fluorescence spectroscopy. The ALA treated cells were irradiated with light at 405 nm with and without subsequent incubation in GSH. Cell survival was measured by colony forming ability. RESULTS: Incubation of cells in growth medium with ALA led to increased synthesis of protoporphyrins in cells which saturated beyond 4 h. The level of protoporphyrin synthesis increased significantly when ALA treated cells were subsequently incubated with GSH in PBS for 15 min. Irradiation of cells incubated with ALA alone led to weak inactivation. However, substantial cell death was observed in ALA treated cells irradiated in the presence of 15 mM GSH. INTERPRETATION & CONCLUSION: Photodynamic inactivation of P. aeruginosa by ALA induced porphyrins can be enhanced if ALA treated cells are further incubated with GSH and irradiated using 405 nm light. These findings may be useful for inactivation of antibiotic resistant strains of P. aeruginosa causing burn and wound infections in hospitalized patients.

Virus papiloma humano: oncogénesis y avances terapéuticos / Human papillomavirus: oncogenesis and therapeutic advances

Los virus Papiloma Humano (HPV), abarcan más de cien subtipos de virus distintos. De ellos, muchos están involucrados en diversas patologías epiteliales benignas y malignas de piel y mucosas, como por ejemplo, el Carcinoma Espinocelular, la enfermedad de Bowen y numerosos tumores de la mucosa genitoanal. Los mecanismos patogénicos involucrados en la tumorogénosis, están en pleno conocimiento. La interacción de las proteínas virales E6 y E7, con las proteínas humanas P53 y la proteína del gen del retenoblastoma, juegan un rol fundamental. Esta revisión pretende poner al día, en cuanto a las lesiones asociadas al HPV, sus mecanismos patogénicos, y las nuevas terapias en desarrollo

In vitro study of biosynthesis of protoporphyrin IX induced by Ù-Aminolevulinic acid in normal and cancerous cells of the human cervix

Bxkground. Ù-Aminolevulinic acid (ALA) is recognized as the starter in the biosynthesis of the heme group, the structural basis of cytochromes, chlorophylls, biliary pigments, and other porphyrins. It is the first intermediary in the biosynthesis of protoporphyrin IX (PpIX), and of ther heme group. PpIX is present in low concentration in normal cells, and in high concentration in tumor cells. Methods. The accumulation of protoporhyrin IX (PpIX) induced by Ù-aminolevulinic acid (ALA) was tested in two cervico-uterine cancer cell lines (HeLa and CaLo), and in normal human cervical epithelial (NHCE) cells. Results. The optimal concentration of ALA that induced maximum levels of intra- and extracellular accumulation of PpIX in both HeLa and NHCE cells was 300 µg of ALA/mL, and for CaLo cells, 150 µg/mL. The viability of HeLa, CaLo, and NHCE cells exposed to ALA measured 81, 98 and 84 percent, respectively. The optimale time for accumulation of PpIX, both intra- and extracellular, was 4 h for HeLa and NHCE cells and 5 h for CaLo cells per 24 h of expusure to optimal concentrations of ALA. After the maximum level of PpIX accumulation was reached, there was a gradual decrease until there was only a small quantity. A statistically significant difference (p <0.0001) was foundf in the accumulation of PpIX, depending on the concentrations of ALA used as well as between cervical cancer cell lines and 1:7, and for NHCE and CaLo cells. 1:5. Conclusions. These results are important for determining the usefulness of the sensitizer (PpIX)

Envolvimento do ácido 5-aminolevulínico como espécie pro-oxidante no plumbismo e envelhecimento / Involvement of the 5-aminolevulinic acid as a pro-oxidant specie in plumbism and the aging process

O Saturnismo e a PAI representam porfirias relacionadas ao acúmulo de ALA, como resultado da inibiçäo de várias enzimas da via biossintética do heme pelo chumbo e defeito genético na biossíntese da PBG-desminase, respectivamente. Bechara et al (1993 - artigo de revisäo) propöem a auto-oxidaçäo da forma enólica do ALA catalisada por complexos de ferro, gerando espécies reativas de oxigênio e o radical ALA, como processo disparador dos sintomas observados nessas patologias. O efeito deletério destas espécies sobre importantes macromoléculas (proteínas, DNA) e estruturas celulares (mitocôndria, retículo endoplasmático) foi evidenciado em vários estudos in vivo e in vitro. O objetivo desta tese é estudar o envolvimento de ALA como espécie pro-oxidante no saturnismo e no processo de envelhecimento. Para tanto, foi necessário o desenvolvimento de um método de dosagem do ALA, pois os métodos até entäo descritos na literatura embora dotados de sensibilidade adequada, eram extremamente trabalhosos e lentos devido ao elevado número de etapas envolvidas na preparaçäo da amostra. O método aqui descrito utiliza a cromatografia em fase reversa em HPLC acoplado a um detector eletroquímico e a derivatizaçäo pré-coluna do ALA com omicron-ftalaldeído (OPA) a um produto indólico. De posse de um método simples e seletivo para a quantificaçäo do ALA, o passo seguinte foi analisar a correlaçäo dos níveis do ALA em plasma com os de chumbo eritrocitário, protoporfirina IX eritrocitária, superóxido dismutase eritrocitária, metehemoglobina (sangue) e quimioluminescência (urina) em amostras coletadas de indivíduos expostos ocupacionalmente ao chumbo. Os resultados obtidos indicaram um grau de correlaçäo acentuado entre vários dos parâmetros testados reforçando o papel do ALA como espécie pró-oxidante de relevância na etiologia do saturnismo. Além disso, foram determinados os níveis de ALA em plasma, fígado e cérebro de ratos caracterizados como adultos jovens (6-8 meses) e velhos (24 meses) e sua correlaçäo com parâmetros indicativos do status oxidativo destes animais. Foi observado um aumento significativo do ALA nos ratos velhos tanto em plasma como em fígado, sendo que o mesmo apresentou uma correlaçäo positiva em fígado e cérebro com ferro näo hemínico, carbonil proteína e "thiobarbituric acid reactive substances" (TBARS). Esses dados nos permitem inferir ao ALA um papel pró-oxidante no processo de envelhecimento

Oxidative stress in acute intermittent porphyria and lead poisoning may be triggered by 5-aminolevulinic acid

Highly reactive oxyradicals can be generated in vitro by iron-catalyzed aerobic oxidation of synthetic and naturally occuring substances capable of enolization in aqueous medium. Of biological interest are alfa-hydroxy- and alfa-aminocarbonyls such as carbohydrates, 5-aminolevulinic acid, and aminoacetone which tautomerize to the corresponding enediols and enolamines and yield oxyradicals initiated by electron transfer to dioxygen. Free radicals have been implicated in several normal and pathological processes. We briefly review our hypothesis of an in vivo prooxidant role of 5-aminolev-ulinic acid (ALA), the heme precursor accumulated in several porphyric disorders (e.g., lead poisoning, acute intermittent porphyria (AIP), tyrosinosis). Accordingly, i) ALA undergoes transition metal-catalyzed oxidation to give O-2, H2O2 and HO; ii) ALA induces iron release from ferritin, lipid peroxidation of cardiolipin-rich vesicles, single strand breaks in plasmid DNA, and guanosine oxidation in calf thymus DNA; iii) ALA causes Ca2+ -mediated rat liver mitochondria permeabilization; iv) rats chronically treated with ALA exhibit increased glycolytic metabolism; v) brain extracts of ALA-treated rats reveal increased levels of thiobarbituric acid reactive substances, direct chemiluminescence intensity, carbonyl proteins, ferritin, and "free iron"and gama-aminobutyric acid-receptor dissociation constant, and vi) patients with AIP and lead-exposed workers present augmented erythrocytic levels of the antioxidant enzymes superoxide dismutase and glutathione peroxidase. These data indicate the involvement of ALA-generated reactive species in the clinical manifestations (neuropathy, mental changes, muscle weakness, hepatoma) shared by the aforementioned inherited and acquired porphyric diseases.

Release of aldehydic products from glutamate or deoxyuridine by delta-aminolevulinic acid in presence of copper ions.

Glutamate or deoxyuridine in the presence of Cu2+ and delta-aminolevulinic acid (ALA) reaction resulted in the formation of thiobarbituric acid reactive compounds (TBAR), which was linear up to 2 hr. TBAR increased linearly with the increase of both ALA and copper ion concentration. Oxygen consumption was directly proportional to the concentration of formaldehyde perhaps formed by secondary oxidation of dimethyl sulfoxide. Addition of oxyradical scavengers in the reaction mixture resulted in the inhibition of TBAR formation.

Acido delta aminolevulínico dehidrasa en pacientes en programa de hemodiálisis / Delta aminolevulinic acid dehydrase in hemodialysis patients

Pacientes insuficientes renales crónicos sometidos a programa de hemodiálisis, pueden presentar un síndrome similar al observado en la porfiria cutánea tarda, que se deberían a fallas en la síntesis de las porfirias por alteraciones metabólicas que acompañan el compromiso renal. En 49 pacientes sometidos a hemodiálisis se encontró la actividad de la ácido delta-aminolevulínico dehidrasa (ALA-d) de glóbulos rojos significativamente disminuida respecto del valor normal (2,18 + - 0,82 vs. 3,10 + - 1,36 U/mg protína). La presencia de Zn+2 10-4 M restablecela actividad del ALA-d a valores normales. Plasma de pacientes hemodializados en el medio de incubación de la ALA-d de glóbulos rojos normales no produce inhibición significativa de la actividad enzimática. La concentración sanguínea de glutación (GSH) se encontró disminuida en los hemodializados respecto de los normales (4,6 + - 2.1 vs. 5,8 + - 1.7 umol/ml sangre), existiendo correlación (p<0,01) entre GSH y la actividad de la ALA-d en glóbulos rojos. El plomo en sangre en la población hemodializada estudiada (0,001 a 0,013 mg/100 g) estaba dentro de los valores normales (hasta 0,30 mg/100 g), lo que se descarta contaminación por este metal en los hemodializados. Los bajos niveles de GSH y de Zn+2 explicarían la disminución de actividad de la ALA-d en glóbulos rojos de pacientes en programa de hemodiálisis