Mitochondrial ATPase activity and membrane fluidity changes in rat liver in response to intoxication with Buckthorn (Karwinskia humboldtiana)

BACKGROUND: Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1,1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment. RESULTS: Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase. CONCLUSIONS: The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.

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 .

Manual and Rotary Instrumentation Ability to Reduce Enterococcus faecalis Associated with Photodynamic Therapy in Deciduous Molars

This aim of this study was to assess the ability of manual or rotary instrumentation associated with photodynamic therapy (PDT) to reduce Enterococcus faecalis using three combinations of light/photosensitizers: toluidine blue O/laser, fuchsin/halogen light and fuchsin/LED. Twenty deciduous molars were selected and contaminated with Enterococcus faecalis (McFarland 0.5 scale). Working length determination was performed by visual method. The teeth were randomly divided into two groups: G1 (n=10): manual instrumentation (Kerr-type files) and G2 (n=10): rotary instrumentation (ProTaper system). The bacteria were collected three times using sterile paper cones compatible with the anatomic diameter of the root canal for 30 s before and after instrumentation and after PDT. The samples were diluted in peptone water, seeded on blood agar plates and incubated in an oven at 37 °C for colony-forming units counting. The decrease of E. faecalis counts after instrumentation and after PDT was compared using the Wilcoxon test, t-test and Kruskal Wallis test. A significant reduction of E. faecalis occurred after manual and rotary instrumentation and after PDT using the three combinations of light/photosensitizer (p<0.05). It may be concluded that both rotary and manual instrumentation reduced E. faecalis. Fuchsin with halogen light or LED irradiation and toluidine blue O with laser irradiation can be used to reduce E. faecalis in root canals of primary molars. PDT can be used as an adjuvant to conventional endodontic treatment.

O objetivo do presente estudo foi avaliar a redução de Enterococcus faecalis após instrumentação manual ou rotatória associada à terapia fotodinâmica (PDT) utilizando 3 combinações luz/fotossensibilizante: azul de toluidina O/laser, fucsina/luz halógena e fucsina/LED. Foram selecionados 20 molares decíduos que foram contaminados com Enterococcus faecalis (escala 0,5 de McFarland). A odontometria foi feita através do método visual. Os dentes foram divididos aleatoriamente em dois grupos: G1 (n=10): instrumentação manual (limas tipo Kerr) e G2 (n=10): instrumentação rotatória (sistema ProTaper). Foram realizadas coletas com cone de papel estéril compatível com o diâmetro anatômico do canal durante 30 s antes e após a instrumentação e a PDT. As amostras foram diluídas em água peptonada, semeadas em placas de agar-sangue e incubadas em estufa a 37 °C para contagem das unidades formadoras de colônias. As comparações antes da redução de E. faecalis após a instrumentação e após a realização da PDT foram realizadas pelo teste de Wilcoxon, teste t e Kruskal Wallis. Houve redução significante de E. faecalis após a instrumentação manual ou rotatória e após realização da PDT com as três combinações de luz/fotossensibilizante (p<0,05). Pode-se concluir que a instrumentação rotatória e manual acarretou a redução de E. faecalis. A fucsina irradiada com luz halógena ou led e o azul de toluidina irradiado com laser podem ser utilizados para redução de E. faecalis do sistema de canais radiculares de molares decíduos. A terapia fotodinâmica pode ser utilizada como coadjuvante ao tratamento endodôntico convencional.

Free radical induced damages to rat liver subcellular organelles: inhibition by Andrographis paniculata extract.

Aqueous extract of Andrographis paniculata was examined for antioxidant activity using rat liver subcellular organelles as model systems. The study deals with two important biological oxidative agents, ascorbate-Fe(+2) and AAPH generating hydroxyl and peroxyl radical, respectively. Oxidative damage was examined against the inhibition of membrane peroxidation, protein oxidation and restoration in decreased SOD and catalase activity. The antimutagenic activity of Ap was examined following inhibition in AAPH induced strand breaks in plasmid pBR322 DNA. Extract was a potent scavenger of DPPH, ABTS radicals, exemplified by ESR signals, O2-*, *OH and H2O2, displayed excellent reducing power, FRAP potentials to reduce Fe (III) --> Fe (II) and had considerable amount of phenolics/ flavonoids contents, an effective antioxidant index. The observed antioxidant effect might be primarily due to its high scavenging ability for ROS. Effect was confirmed ex vivo following inhibition in peroxidation, restoration in SOD enzyme, SOD band intensity and protein degradation in Ap fed liver homogenate. Based on these results, it was concluded that the aqueous extract of Andrographis paniculata might emerge as a potent antiradical agent against various pathophysiological oxidants.

Odontologic use of copper/aluminum alloys: mitochondrial respiration as sensitive parameter of biocompatibility

Copper/aluminum alloys are largely utilized in odontological restorations because they are less expensive than gold or platinum. However, tarnishing and important corrosion in intrabuccal prostheses made with copper/aluminum alloys after 28 days of use have been reported. Several kinds of food and beverage may attack and corrode these alloys. Copper is an essential component of several important enzymes directly involved in mitochondrial respiratory metabolism. Aluminum, in contrast, is very toxic and, when absorbed, plasma values as small as 1.65 to 21.55 µg/dl can cause severe lesions to the nervous system, kidneys, and bone marrow. Because mitochondria are extremely sensitive to minimal variation of cellular physiology, the direct relationship between the mitocondrial respiratory chain and cell lesions has been used as a sensitive parameter to evaluate cellular aggression by external agents. This work consisted in the polarographic study of mitochondrial respiratory metabolism of livers and kidneys of rabbits with femoral implants of titanium or copper/aluminum alloy screws. The experimental results obtained did not show physiological modifications of hepatic or renal mitochondria isolated from animals of the three experimental groups, which indicate good biocompatibility of copper/aluminum alloys and suggest their odontological use

Calcium induced alterations in structural and functional role of phospholipids in Microsporum gypseum.

The effect of calcium on the structural and functional aspects of phospholipids in Microsporum gypseum was examined. Cells grown in presence of calcium exhibited increased content of phospholipids and enhanced synthesis of phospholipids as monitored by the incorporation of [32P] orthophosphoric acid. The rise in the levels of phospholipids was found to be due to increased synthesis of fatty acids as observed from [14C] acetate incorporation studies. The rise in the levels of phospholipids were reflected in the subcellular fractions also. Change in the phospholipid composition increased the fluidity of the membrane as is evident from fluorescence polarization studies using 1-anilinonaphthalene-8-sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The increased membrane fluidity was consistent with the enhanced uptake of [3H] proline in calcium grown cells.