Phototoxic effect of curcumin on methicillin-resistant Staphylococcus aureus and L929 fibroblasts.

Photodynamic therapy has been investigated as an alternative method of killing pathogens in response to the multiantibiotic resistance problem. This study evaluated the photodynamic effect of curcumin on methicillin-resistant Staphylococcus aureus (MRSA) compared to susceptible S. aureus (MSSA) and L929 fibroblasts. Suspensions of MSSA and MRSA were treated with different concentrations of curcumin and exposed to light-emitting diode (LED). Serial dilutions were obtained from each sample, and colony counts were quantified. For fibroblasts, the cell viability subsequent to the curcumin-mediated photodynamic therapy was evaluated using the MTT assay and morphological changes were assessed by SEM analysis. Curcumin concentrations ranging from 5.0 to 20.0 µM in combination with any tested LED fluences resulted in photokilling of MSSA. However, only the 20.0 µM concentration in combination with highest fluence resulted in photokilling of MRSA. This combination also promoted an 80% reduction in fibroblast cell metabolism and morphological changes were present, indicating that cell membrane was the main target of this phototherapy. The combination of curcumin with LED light caused photokilling of both S. aureus strains and may represent an alternative treatment for eradicating MRSA, responsible for significantly higher morbidity and mortality and increased healthcare costs in institutions and hospitals.

Determination of rupatadine in pharmaceutical formulations by a validated stability-indicating MEKC method.

A stability-indicating MEKC was developed and validated for the analysis of rupatadine in tablet dosage forms, using nimesulide as internal standard. The MEKC method was performed on a fused-silica capillary (50 microm id; effective length, 40 cm). The BGE consisted of 15 mM borate buffer and 25 mM anionic detergent SDS solution at pH 10. The capillary temperature was maintained at 35 degrees C and the applied voltage was 25 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 5 s, with detection by photodiode array detector set at 205 nm. The method was linear in the range of 0.5-150 microg/mL (r2=0.9996). The specificity and stability-indicating capability of the method were proven through degradation studies inclusive by MS, and showing also that there was no interference of the excipients and no increase of the cytotoxicity. The accuracy was 99.98% with bias lower than 1.06%. The LOD and LOQ were 0.1 and 0.5 microg/mL, respectively. The proposed method was successfully applied for the quantitative analysis of rupatadine in pharmaceutical formulations, and the results were compared to a validated RP-LC method, showing non-significant difference (p>0.05).

Citotoxicidad de un sellador experimental a base de trióxido mineral / Citotoxicity of a mineral trioxide based experimental root canal sealer

El objetivo de este estudio fue determinar la citotoxicidad no específica de un sellador endodóntico experimental a base del polvo de ProRoot (MTA) mezclado con una resina polivinílica de base acuosa como vehículo en reemplazo del agua destilada, comparándolo con un material original, mediante la técnica de extendido en agar. Para el estudio se utilizaron 18 placas de Petri conteniendo las células L-929 en agar estéril, a las que se les adicionó solución de rojo neutro. Muestras preparadas de cemento Pro Root MTA y polvo de Pro Root MTA + resina polivinílica fueron colocadas en el interior de anillos circulares de silicona. Los controles positivos consistieron en los mismos anillos de silicona con hidróxido de Na al 10 por ciento y para los controles negativos se utilizaron los anillos vacíos. Todos los anillos fueron esterilizados con luz ultravioleta. Se colocaron 4 anillos por placa, conniendo los materiales a estudiar junto con los controles y se incubaron durante 24 hs en estufa gaseada a 37ºC en atmósfera de CO2, al 5 por ciento saturada de humedad. Las placas se examinaron utilizando un microscopio de óptica invertida con ocular micrométrico y se determinó el índice de decoloración y de lisis para cada especimen. Los resultados obtenidos indicaron que las muestras de Pro Root y polvo de Pro Root con una resina polivinílica presentaron el mismo nivel de citotoxicidad, ya que el área de decoloración y lisis se circunscribió a la superficie que se encontraba justo por debajo del material ensayado. En el caso del control positivo (hidróxido de sodio) el halo de decoloración y lisis superó los 6mm desde el material ensayado. El control negativo no presentó decoloración ni lisis, aún debajo de la superficie del mismo. A la luz de los resultados obtenidos con la metodología utilizada podemos concluir que el uso de una emulsión acuosa de alcoholes polivinílicos mezclado con polvo de Pro Root (MTA) no altera la citotoxicidad del material original.(AU)

Cytotoxicity of hard chairside reline resins: effect of microwave irradiation and water bath postpolymerization treatments.

PURPOSE: To evaluate the influence of water bath and microwave postpolymerization treatments on the cytotoxicity of 6 hard reline acrylic resins. MATERIALS AND METHODS: The materials tested were Tokuso Rebase Fast (TR), Ufi Gel Hard (UGH), Duraliner II (D), Kooliner (K), New Truliner (NT), and Light Liner (LL). LL resin was additionally tested with an air-barrier coating (LLABC). Nine disks of each material (10 x 1 mm) were made and divided into 3 groups: group 1 (no postpolymerization treatment); group 2 (postpolymerization in microwave oven); group 3 (postpolymerization in water bath at 55 degrees C for 10 minutes). L929 cells were cultured in 96-well plates and incubated for 24 hours in Eagle's medium. Eluates prepared from the disks or medium without disks (control) replaced the medium. Cytotoxicity was assessed by both dehydrogenase succinic activity (MTT) assay and incorporation of radioactive 3H-thymidine assay. Tests were carried out in quadruplicate and repeated twice. Differences between groups were determined by analysis of variance with Tukey multiple-comparison intervals (alpha = .05). RESULTS: For MTT assay, the postpolymerization treatments had no effect on the cytotoxicity of all materials (P > .05). For 3H-thymidine assay, the postpolymerization treatments significantly decreased the cytotoxicity of UGH (P < .05). The cytotoxicity of K, NT, LL, and LLABC increased after microwave irradiation (P < .05). TR, NT, and LLABC showed an increase in cytotoxicity after water bath (P < .05). CONCLUSION: When assessed by MTT assay, the cytotoxicity of the materials was not affected by postpolymerization treatments. 3H-Thymidine assay showed that the cytotoxicity of the resins was not improved by the postpolymerization treatments, with the exception of UGH.

Effects of FSH and 17beta-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells.

In the present study, we analyzed human follicle-stimulating hormone (FSH)-induced cell proliferation and transactivation of estrogen-sensitive reporter genes-in L cells stably expressing the human FSH receptor [L-(hFSHR(+)) cells]. In order to dissect the signaling pathways involved in this process, L-(hFSHR(+)) cells were transiently transfected with either the 3X-ERE-TAT-Luc or the ERE-VitA2-TK-CAT reporter genes and treated with FSH or PKA activators (cholera toxin, forskolin and 8-Br-cAMP) in the presence or absence of various kinase inhibitors. We found that FSH and all PKA activators, specifically induced transactivation of both reporter genes. Transactivation of estrogen-sensitive genes by FSH or PKA activators were blocked (approximately 90%) by H89 (PKA inhibitor) and LY294002 but not by Wortmannin (PI3-K inhibitors), 4-OH-tamoxifen, ICI182,780 or SB203580 (p38 MAPK inhibitor); PD98059 (ERK1/2 inhibitor) partially (approximately 30%) blocked the FSH-mediated effect. The combination of FSH and estradiol resulted in a synergistic effect on transactivation as well as on cell proliferation, and this enhancement was attenuated by antiestrogens. We additionally analyzed the participation of the coactivators SRC-1 and cAMP response element binding protein (CREB)-binding protein (CBP) in FSH-evoked estrogen receptor (ER)-dependent transactivation; we found that CBP but not SRC-1 potentiated FSH-induced transcriptional activation of both ER-sensitive reporters, being this effect stronger on the ERE-VitA2-TK-CAT than on the 3X-ERE-TAT-Luc reporter. Thus, in L-(hFSHR(+)) cells FSH induces transcriptional activation of estrogen-sensitive genes through an A-kinase-triggered signaling pathway, using also to a lesser extent the ERK1/2 and p38 pathways. PI3-K is not apparently involved in this FSH-mediated process since LY294002, but not Wortmannin, specifically binds ERs and completely blocks estrogen action. Presumably, CBP cooperates with the ER on genes that contain estrogen responsive elements through mechanisms involving the participation of other proteins and/or basal transcription factors (e.g. CREB), which in turn mediate the transcriptional response of estrogen-sensitive reporter genes to FSH stimulation.

Cytotoxicity of denture base resins: effect of water bath and microwave postpolymerization heat treatments.

PURPOSE: This study compared the effect of two postpolymerization heat treatments on the cytotoxicity of three denture base resins on L929 cells using 3H-thymidine incorporation and MTT assays. MATERIALS AND METHODS: Sample disks of Lucitone 550, QC 20, and Acron MC resins were fabricated under aseptic conditions and stored in distilled water at 37 degrees C for 48 hours. Specimens were then divided into three groups: (1) heat treated in microwave oven for 3 minutes at 500 W; (2) heat treated in water bath at 55 degrees C for 60 minutes; and (3) no heat treatment. Eluates were prepared by placing three disks into a sterile glass vial with 9 mL of Eagle's medium and incubating at 37 degrees C for 24 hours. The cytotoxic effect from the eluates was evaluated using the 3H-thymidine incorporation and MTT assays, which reflect DNA synthesis levels and cell metabolism, respectively. RESULTS: The components leached from the resins were cytotoxic to L929 cells when 3H-thymidine incorporation assay was employed. In contrast, eluates from all resins revealed noncytotoxic effects as measured by MTT assay. For both MTT assay and 3H-thymidine incorporation, the heat treatments did not decrease the cytotoxicity of the materials tested. CONCLUSION: Resins were graded by 3H-thymidine incorporation assay as slightly cytotoxic and by MTT assay as noncytotoxic. Cytotoxicity of the denture base materials was not influenced by microwave or water bath heat treatment.

Cytotoxicity of some sesquiterpene lactones "in vitro".

The cytotoxic effects of sesquiterpene gamma-lactones in tissue culture were studied on two different cell lines, one of them fibroblastoid cells L-929 from mice areolar tissue, and the other epithelial cells HEp-2 from human laryngeal carcinoma. Ten lactones were tested, all of them having in their molecule an alpha methylene gamma lactone group, which had been described as conferring cytotoxic activity. Five of these lactones have a furanic ring in their molecule whose presence gives them a higher cytotoxic activity. Different doses of each lactone were tested. Cell number and exclusion tests were simultaneously performed per dose, obtaining for all of them the ED50, which was the calculated as the effective dose inhibiting growth to 50 per cent of control growth. The ED50 was about 1 to 5 microgram in those lactones with furanic ring. The two cell lines had a different response to the same lactone.