M. Celeste Lopes

Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia Magna

In this work we investigated the possibility of using the enzyme acetylcholinesterase (AChE) activity in Daphnia magna homogenates, both in vivo and in vitro conditions, as a specific method for rapid toxicity evaluations. The results from in vivo and in vitro AChE inhibition tests were compared with 48 hours EC50 values obtained in conventional acute bioassays. EC50 values from in vivo AChE inhibition tests were: 2.4 micrograms/l for parathion, 0.2 microgram/l for paraoxon; DCA and cadmium at the concentrations tested had no effects on enzyme activity. I50 values were 764 micrograms/l for parathion, 0.08 micrograms/l for paraoxon and 3367 micrograms/l for cadmium; DCA did not affect AChE activity measured in in vitro conditions. EC50 values from conventional acute tests were: 2.2 micrograms/l for parathion, 0.2 microgram/l for paraoxon, 163 micrograms/l for DCA and 9.5 micrograms/l for cadmium. Our results indicated that the in vivo AChE inhibition test is selective, being very sensitive to detect toxicity of the organophosphates tested. The in vitro AChE inhibition assay is less time consuming, requires less human effort and produces less toxic waste than conventional acute bioassays and the in vivo AChE inhibition test. However, it does not take into account the effect of the metabolization of the toxicants inside live organisms; since the organophosphate metabolism may be activative or degradative, the toxic potential of the parent compound may be under or over evaluated in in vitro conditions.

Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-KB activation and iNOS expression in bovine articular chondrocytes

Our previous studies showed that reactive oxygen species (ROS) are required for the pro‐inflammatory cytokine interleukin‐1β (IL‐1) to induce the activity of the Nuclear transcription Factor‐kappaB (NF‐κB) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H2O2) and the superoxide radical, two major ROS, in mediating those IL‐1‐induced responses. The results obtained show that chondrocytes produce both H2O2 and superoxide radical in response to IL‐1. Treatment of the chondrocyte cultures with H2O2 alone did not induce NF‐κB activation or iNOS expression. Addition of H2O2 simultaneously with IL‐1 did neither enhance nor inhibit NF‐κB activation and iNOS expression, relatively to treatment with IL‐1 alone. Accordingly, treatment with catalase did not inhibit those IL‐1‐induced responses. Treatment with superoxide dismutase, however, effectively prevented IL‐1‐induced IκB‐α degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL‐1‐induced IκB‐α degradation and the consequent NF‐κB activation and iNOS expression in chondrocytes, whereas H2O2 does not seem to participate in those IL‐1‐induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL‐1‐induced signaling pathway that leads to NF‐κB activation and to the expression of NF‐κB‐dependent genes in bovine articular chondrocytes.

Anti-Inflammatory Activity of Cymbopogon citratus Leaf Infusion in Lipopolysaccharide-Stimulated Dendritic Cells: Contribution of the Polyphenols

Cymbopogon citratus, an herb known worldwide as lemongrass, is widely consumed as an aromatic drink, and its fresh and dried leaves are currently used in traditional cuisine. However, little is known about the mechanism of action of C. citratus, namely, the anti-inflammatory effects of its dietary components. Because nitric oxide (NO), produced in large quantities by activated inflammatory cells, has been demonstrated to be involved in the pathogenesis of acute and chronic inflammation, we evaluated the effects of the infusion of dried leaves from C. citratus, as well as its polyphenolic fractions--flavonoid-, tannin-, and phenolic acid-rich fractions (FF, TF, and PAF, respectively)--on the NO production induced by lipopolysaccharide (LPS) in a skin-derived dendritic cell line (FSDC). C. citratus infusion significantly inhibited the LPS-induced NO production and inducible NO synthase (iNOS) protein expression. All the polyphenolic fractions tested also reduced the iNOS protein levels and NO production stimulated by LPS in FSDC cells, without affecting cell viability, with the strongest effects being observed for the fractions with mono- and polymeric flavonoids (FF and TF, respectively). Our results also indicated that the anti-inflammatory properties of FF are mainly due to luteolin glycosides. In conclusion, C. citratus has NO scavenging activity and inhibits iNOS expression and should be explored for the treatment of inflammatory diseases, in particular of the gastrointestinal tract.

Role of oxidative stress in ERK and p38 MAPK activation induced by the chemical sensitizer DNFB in a fetal skin dendritic cell line

The intracellular mechanisms involved in the early phase of dendritic cell (DC) activation upon contact with chemical sensitizers are not well known. The strong skin sensitizer 2,4‐dinitrofluorobenzene (DNFB) was shown to induce the activation of mitogen‐activated protein kinases (MAPK) in DC. In the present study, we investigated a putative role for oxidative stress in DNFB‐induced MAPK activation and upregulation of the costimulatory molecule CD40. In a DC line generated from fetal mouse skin, DNFB induced a significant increase in protein oxidation, measured by the formation of carbonyl groups, while it had almost no effect on lipid peroxidation. The antioxidants glutathione and vitamin E, which inhibit protein and lipid oxidation, respectively, were used to assess the role of oxidative stress in DNFB‐induced MAPK activation. Glutathione, but not vitamin E, inhibited DNFB‐induced p38 MAPK and ERK1/2 phosphorylation, whereas none of the antioxidants interfered significantly with the DNFB‐induced upregulation of CD40 protein levels. Taken together, these results indicate that DNFB activates p38 MAPK and ERK1/2 via production of reactive oxygen species, and that protein oxidation plays an important role in MAPK activation.

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.

Granulocyte-macrophage colony-stimulating factor activates the transcription of nuclear factor kappa B and induces the expression of nitric oxide synthase in a skin dendritic cell line

Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM‐CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal‐skin‐derived dendritic cell line (FSDC) and, consequently, increases the nitrite production from 11.9 ± 3.2 μmol/L (basal level) to 26.9 ± 4.2 μmol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC50) of 19.3 μmol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM‐CSF induces the phosphorylation and degradation of the inhibitor of NF‐κB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF‐κB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM‐CSF activates the transcription factor NF‐κB. Together, these results show that GM‐CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF‐κB pathway.

Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell line

Abstract:  Nuclear factor kappa B (NF‐kB) and activating protein‐1 (AP‐1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin‐derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO4) and increases the expression of the iNOS protein, as determined by immunofluorescence and Western blot analysis. The sensitizer NiSO4 increased cytoplasmic iNOS expression by 31.9 ± 10.3% and nitrite production, as assayed by the Griess reaction, by 27.6 ± 9.5%. Electrophoretic mobility shift assay (EMSA), showed that 30 min of FSDC exposure to NiSO4 activates the transcription factor NF‐kB by 58.2 ± 7.0% and 2 h of FSDC exposure to NiSO4 activates the transcription factor AP‐1 by 26.0 ± 1.4%. Together, these results indicate that NiSO4 activates the NF‐kB and AP‐1 pathways and induces iNOS expression in skin dendritic cells.

Comparative study between the toxicity of 3,4-dichloroaniline and sodium bromide with 21-days chronic test and using lactate dehydrogenase activity of Daphnia magna Straus

Abstract We studied the effect of 3,4-dichloroaniline (DCA) and sodium bromide (NaBr) on the lactate dehydrogenase (LDH) activity in homogenate of Daphnia magna Straus cultured during 21 days in the presence of several concentrations of each chemical. The activity of LDH increased at concentrations of 10 μg/l of DCA and 19 mg/l of NaBr. These concentrations correspond to LOEC values obtained in 21-day chronic tests with Daphnia magna, which suggests that LDH activity may be used as a faster indicator of toxicity in tests with D. magna.

Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-kappaB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line.

AIMS: Nitric oxide (NO) has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in a mouse fetal skin dendritic cell line. METHODS: NO production was assessed by the method of Griess. Expression of the inducible isoform of nitric oxide synthase (iNOS) protein was evaluated by western blot analysis and immunofluorescence microscopy. Western blot analysis was also performed to evaluate cytosolic IkappaB-alpha (IkappaB-alpha) protein levels. The electrophoretic mobility shift assay was used to evaluate the activation or inhibition of nuclear factor kappa B (NF-kappaB). RESULTS: GM-CSF induced iNOS expression and NO production, and activated the transcription factor NF-kappaB. Dexamethasone inhibited, in a dose-dependent manner, NO production induced by GM-CSF. Addition of dexamethasone to the culture, 30 min before GM-CSF stimulation, significantly inhibited the cellular expression of iNOS. Dexamethasone also inhibited GM-CSF-induced NF-kappaB activation by preventing a significant decrease on the IkappaB-alpha protein levels, thus blocking NF-kappaB migration to the nucleus. CONCLUSIONS: The corticosteroid dexamethasone inhibits GM-CSF-induced NF-kappaB activation, iNOS protein expression and NO production. These results suggest that dexamethasone is a potent inhibitor of intracellular events that are involved on NO synthesis, in skin dendritic cells.

Action of antiestrogens on the (Ca2+, +Mg2+)- ATPase and Na+/Ca2+ exchange of brain cortex membranes

The effect of tamoxifen (TAM) and other antiestrogens on the Ca2+ transport activity of synaptic plasma membranes (SPM) and microsomal membranes isolated from sheep brain cortex was investigated. The maximal (Ca2+ + Mg2+)-ATPase activity of SPM, which is reached at a pCa of about 6.0-6.5, is decreased by about 30% in the presence of 50 microM TAM, whereas the (Ca2+ + Mg2+)-ATPase activity of microsomes, which is maximal at a pCa of about 5.0, is decreased by about 90% by 50 microM TAM. In parallel experiments, we observed that the ATP-dependent Ca2+ uptake is also affected differently by TAM in the two membrane preparations. We found that 50 microM TAM inhibits SPM Ca2+ uptake by about 25-30%, whereas the ATP-dependent Ca2+ uptake by the microsomal fraction is inhibited by about 60%. No significant effect of TAM was observed on the Na+/Ca2+ exchange of either membrane system. The results indicate that TAM is a more potent inhibitor of the active, calmodulin-independent Ca2+ transport system of the intracellular membranes than of that of the plasma membranes, which is calmodulin-dependent. It appears that TAM inhibits calmodulin-mediated reactions, probably through its binding to calmodulin, as we showed previously. However, the Ca2+ transport system of microsomes, which does not depend on calmodulin, is also particularly sensitive to TAM.