Indium chloride-induced micronuclei via reactive oxygen species in Chinese hamster lung fibroblast V79 cells.

We study the cytotoxicity of indium chloride (InCl3) in Chinese hamster lung fibroblasts, the V79 cells, using MTT assay. The results showed that InCl3 did not induce significant cytotoxicity at various concentrations tested. In addition, the frequency of micronuclei (MN) was assayed to evaluate the genotoxic effects of InCl3 in V79 cells. InCl3 at concentrations ranged 0.1-1 µM significantly increased MN frequency in a concentration-dependent manner. Both catalase and superoxide dismutase at concentrations of 75 and 150 µg/mL significantly inhibited InCl3-induced MN. Similarly, Germanium oxide (GeO2) and dimercaprol expressed antigenotoxic effects. From these findings, it is concluded that InCl3 is a potent genotoxic chemical, which may be mediated partly by inducing oxidative stress. The significance of this study shows that the workers in the semiconductor factories should be cautious in exposing to the hazardous genotoxic InCl3.

Evaluation of differential representative values between Chinese hamster cells and human lymphocytes in mitomycin C-induced cytogenetic assays and caspase-3 activity.

Chinese hamster ovary (CHO) cells, its lung fibroblasts (V79), and human lymphocytes are routinely used in in vitro cytogenetic assays, which include micronuclei (MN), sister chromatid exchange (SCE), and chromosome aberration (CA) assays. Mitomycin C (MMC), a DNA cross-link alkylating agent, is both an anticancer medicine and a carcinogen. To study the differential representative values of cell types in MMC-treated cytogenetic assays and its upstream factor, cysteine aspartic acid-specific protease (caspase)-3. Among the chosen cell types, lymphocytes expressed the highest sensitivity in all three MMC-induced assays, whereas CHO and V79 showed varied sensitivity in different assays. In MN assay, the sensitivity of CHO is higher than or equal to V79; in SCE assay, the sensitivity of CHO is the same as V79; and in CA assay, the sensitivity of CHO is higher than V79. In-depth analysis of CA revealed that in chromatid breaks and dicentrics formation, lymphocyte was the most sensitive of all and CHO was more sensitive than V79; and in acentrics and interchanges formation, lymphocyte was much more sensitive than the others. Furthermore, we found caspase-3 activity plays an important role in MMC-induced cytogenetic assays, with MMC-induced caspase-3 activity resulting in more sensitivity in lymphocytes than in CHO and V79. Based on these findings, lymphocyte will make a suitable predictive or representative control reference in cytogenetic assays and caspase-3 activity with its high specificity, positive predictive value, and sensitivity.

Cardioprotective effects of luteolin during ischemia-reperfusion injury in rats.

BACKGROUND: Antioxidants effectively reduce ischemia-reperfusion (IR) injury. The cardioprotective effects of luteolin, a flavonoid that exhibits antioxidant properties and is widely available in many fruits and vegetables, were examined in rats subjected to myocardial IR injury. METHODS AND RESULTS: Rats were subjected to myocardial ischemia or reperfusion injury to evaluate the antiarrhythmic effects of luteolin. Myocardial infarct size was determined histochemically with triphenyltetrazolium chloride staining of the left ventricle. Luteolin was administered intravenously 15min before occlusion of the coronary artery. The incidence and duration of ventricular tachycardia and ventricular fibrillation and mortality during myocardial ischemia were significantly reduced by luteolin (10µg/kg). Similarly, luteolin (1µg/kg) reduced ventricular arrhythmias and mortality during the reperfusion phase. Pretreatment with luteolin decreased plasma lactate dehydrogenase and nitric oxide (NO) levels. Luteolin (10µg/kg) significantly reduced the myocardial infarct size, as well as malondialdehyde production in tissue samples of myocardial IR injury. Luteolin also downregulated inducible NO synthase protein and mRNA expression, but did not significantly alter neuronal NO synthase or endothelial NO synthase expression. CONCLUSIONS: Luteolin is capable of protecting the myocardium against IR injury. The actions of luteolin are at least partly mediated through downregulation of NO production and its own antioxidant properties.

Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils.

AIM: To investigate whether luteolin, the major polyphenolic components of Lonicera japonica, has beneficial effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to determine whether the protective mechanism involves anti-inflammatory effects on neutrophils. METHODS: ALI was induced with intratracheal instillation of LPS in mice. The level of ALI was determined by measuring the cell count and protein content in bronchoalveolar lavage (BAL) fluid. Neutrophils were stimulated with formyl-Met-Leu-Phe (fMLP) or LPS in vitro. Chemotaxis and superoxide anion generation were measured to evaluate neutrophil activation. The potential involvement of intracellular signaling molecules in regulating neutrophil activation was analyzed by using Western blot. RESULTS: LPS induced ALI in mice, as evidenced with leukocyte infiltration and protein leakage into the lungs. Luteolin attenuated LPS-induced leukocyte infiltration and protein extravasation. In cell studies, luteolin attenuated the fMLP-induced neutrophil chemotaxis and respiratory burst (IC(50) 0.2+/-0.1 micromol/L and 2.2+/-0.8 micromol/L, respectively), but had a negligible effect on superoxide anion generation during phorbol myristate acetate stimulation. Furthermore luteolin effectively blocked MAPK/ERK kinase 1/2 (MEK), extracellular signal-regulated kinase (ERK), and Akt phosphorylation in fMLP- and LPS-stimulated neutrophils. CONCLUSION: These results indicate that luteolin has beneficial effects against LPS-induced ALI in mice, and the attenuation of neutrophil chemotaxis and respiratory burst by luteolin involves the blockade of MEK-, ERK-, and Akt-related signaling cascades.

Protective effects of antioxidants on micronuclei induced by irradiated 9-fluorenone/N,N-dimethyl-p-toluidine in CHO cells.

9-Fluorenone (9F), the aromatic photosensitizer, is widely used as an initiator in visible-light (VL) cured resin systems. There is growing concern that 9F may produce genetic damage by inducing mutation. In this study, 9F in the presence or absence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that a dose-related increase in the frequency of MN and prolonged cell cycles in 9F with or without DMT in the presence or absence of VL irradiation (p < 0.05). The rank orders with respect to genotoxicity and cytotoxicity were found to be as follows: 9F/DMT +VL > 9F/DMT = 9F + VL > 9F. To determine whether oxidative stress could modulate MN induced by 9F/DMT with or without VL irradiation in CHO cells, cells were pretreated with N-acetyl-L-cysteine (NAC), ascorbic acid, and alpha-tocopherol. The pretreatment with antioxidants could diminish not only the prolonged cell cycle but also the decreased frequency of MN which is induced by 9F with or without DMT in the presence or absence of VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by 9F in the presence or absence of DMT with or without VL irradiation in CHO cells, indicating clastogenic activity of 9F/DMT in vitro. These antioxidants act as the antagonists against the genotoxicity and cytotoxicity of 9F/DMT. Thus, leaching photoinitiator and reducing agent might be contributing the sources of oxidative stress.

Protective effects of antioxidants on micronuclei induced by camphorquinone/N,N-dimethyl-p-toluidine employing in vitro mammalian test system.

Camphorquinone (CQ) is widely used as an initiator in modern visible-light (VL) cured resin systems. CQ is also characterized as a potential allergenic compound. To date, there is growing concern that CQ may produce genetic damage by inducing mutation. In this study, CQ in the presence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that an increase in the numbers of MN was observed with CQ/DMT with or without VL irradiation (p < 0.05). Significant prolongation of cell cycles was observed by the treatment with CQ/DMT with or without VL irradiation (p < 0.05). In addition, VL irradiated CQ/DMT was found to exhibit significantly genotoxic and cytotoxic effects as compared with CQ/DMT alone (p < 0.05). Furthermore, to determine whether oxidative stress could modulate the MN induced by CQ/DMT with or without VL irradiation in CHO cells, cells were pre-treated with various antioxidants 10 mM N-acetyl-L-cysteine (NAC), 2 mM ascorbic acid, and 2 mM alpha-tocopherol. The pre-treatment with antioxidants could antagonize not only the increased MN cells but also the prolonged cell cycle induced by CQ/DMT with or without VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by CQ/DMT employing mammalian test system, indicating clastogenic activity of CQ/DMT with or without VL irradiation in vitro. In addition, VL irradiated CQ/DMT exhibits higher genotoxic and cytotoxic effects than CQ/DMT alone. Moreover, NAC, ascorbic acid, and alpha-tocopherol act as the antagonists against the genotoxicity and cytotoxicity of CQ/DMT with or without VL irradiation.

Effective attenuation of acute lung injury in vivo and the formyl peptide-induced neutrophil activation in vitro by CYL-26z through the phosphoinositide 3-kinase gamma pathway.

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the polymorphonuclear leukocyte (PMNL) infiltration and protein leakage into the lungs in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as determined on the basis of PMNL and protein contents in bronchoalveolar lavage (BAL) fluid and myeloperoxidase (MPO) content in whole lung extracts. CYL-26z also attenuated the formyl-Met-Leu-Phe (fMLP)-induced neutrophil chemotaxis and respiratory burst in vitro (IC(50) 8.4+/-0.9microM and 2.0+/-0.6microM, respectively). CYL-26z had no effect on superoxide anion (O(2)(-)) generation during dihydroxyfumaric acid autoxidation or on the NADPH oxidase activity in two cell-free systems (the arachidonic acid-induced assembly of NADPH oxidase and the preassembled oxidase caused by phorbol ester treatment), whereas it inhibited NaF-induced respiratory burst. Inhibition of respiratory burst by CYL-26z was readily reversible by washing. Only slight, but significant, inhibition of extracellular signal regulated kinase (ERK) phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation in response to fMLP by CYL-26z up to 30microM was obtained. CYL-26z effectively blocked the formation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) as determined by immunofluorescence microscopy and flow cytometry assays and the dual phosphorylation of protein kinase B (PKB/Akt) on S473 and T308 residues in fMLP-stimulated neutrophils. The membrane recruitment of p110gamma and Ras, the Ras activation, and the association between p110gamma and Ras were also attenuated by CYL-26z. These results indicate that the blockade of Ras activation by CYL-26z attenuated the downstream phosphoinositide 3-kinase (PI3K) gamma signaling, which is involved in chemoattractant-induced neutrophil chemotaxis and respiratory burst, and may have a beneficial anti-inflammatory effect on ALI.

Activation of phosphoinositide 3-kinase and Src family kinase is required for respiratory burst in rat neutrophils stimulated with artocarpol A.

Artocarpol A (ART), a natural product isolated from Artocarpus rigida, stimulated superoxide anion (O2*-) generation, which was inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, in rat neutrophils. ART stimulated phosphorylation of protein kinase B (PKB/Akt) on both T308 and S473 residues, and LY 294002 inhibited these effects. Rat neutrophils expressed both class IA PI3K subunits (p85, p110alpha, p110beta, and p110delta) and a class IB PI3K subunit (p110gamma) as assessed by a combination of Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) approaches. Stimulation of neutrophils with ART evoked phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) formation, which reached a maximal level at 2 min and was attenuated by LY 294002, as evidenced by immunofluorescence microscopy and by flow cytometry. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5, 0.5 and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110gamma immunoprecipitates contain Ras. ART stimulated Src family kinase activation, which was detectable within 1.5 min of incubation with ART. Both Src kinase activity and PtdIns(3,4,5)P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1 analog). PP1 analog also attenuated the ART-stimulated O2*- generation in rat neutrophils. These results indicate that the stimulation of respiratory burst by ART in neutrophils implicates PI3K signaling.

Caffeic acid phenethyl ester ameliorates cerebral infarction in rats subjected to focal cerebral ischemia.

The effects of caffeic acid phenethyl ester (CAPE), an antioxidant derived from propolis, on the infarct volume elicited by focal cerebral ischemia were studied on Long-Evans rats. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of bilateral common carotid arteries (CCA) for 60 min. The rats were sacrificed 24 h later and serial brain slices of 2 mm thickness were taken and stained for the measurement of infarct area. CAPE was administered intravenously 15 min before MCA occlusion. Pretreatment of CAPE (0.1, 1 and 10 microg/kg) significantly reduced the total infarct volume from 169.6 +/- 14.5 mm3 (control) to 61.0 +/- 24.1 mm3 (0.1 microg/kg CAPE), 47.4 +/- 9.1 mm3 (1 microg/kg CAPE), and 42.4 +/- 8.7 mm3 (10 microg/kg CAPE), respectively. Plasma nitric oxide (NO) content was significantly increased in rats subjected to focal cerebral ischemia. It is concluded that CAPE possesses neuroprotective properties in focal cerebral ischemia injury in rats possibly through its antioxidant effect and/or via the upregulation of NO production.

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways.

1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by phenolic (3E)-4-(2-hydroxyphenyl)but-3-en-2-one in RAW 264.7 macrophages.

The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-kappaB (NF-kappaB) DNA binding and NF-kappaB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-kappaBalpha (IkappaBalpha) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH(2)-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-kappaB.

Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation.

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC50 1.2+/-0.2 microM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 microM. CYL-26z blocked the membrane recruitment of protein kinase C-alpha (PKC-alpha) at concentrations of CYL-26z > or =3 microM, but failed to affect the membrane association of PKC-betaI and -betaII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC50 3.8+/-0.8 microM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC50 1.8+/-1.0 microM and 1.8+/-0.9 microM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPgammaS (IC50 1.3+/-0.5 microM). In contrast, the GTPgammaS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 microM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and beta-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.

Antiarrhythmic effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia/reperfusion injury in rats.

OBJECTIVES: The present study was designed to determine the antiarrhythmic effect of caffeic acid phenethyl ester (CAPE), an active component of propolis, which exhibits antioxidant properties, in rats subjected to myocardial ischemia and ischemia-reperfusion (I/R) injury. DESIGN AND METHODS: Rats were subjected to 30 min coronary artery occlusion for evaluating the effect of CAPE on the myocardial ischemia injury. While in the myocardial I/R injury study, the coronary artery was ligated for a 5-min period of ischemia followed by a 30-min period of reperfusion. Animals were pretreated with or without CAPE before coronary artery ligation and the severity of myocardial ischemia- and I/R-induced arrhythmias and mortality were compared. RESULTS: Pretreatment of CAPE (0.1 and 1 microg/kg) not only reduced both the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) but also decreased the mortality during the myocardial ischemia and I/R injury period. CONCLUSIONS: Our results suggest that CAPE is a potent antiarrhythmic agent with cardioprotective effects in myocardial ischemia and I/R injury rats.

Taicatoxin inhibits the calcium-dependent slow motility of mammalian outer hair cells.

The effects of taicatoxin on the slow motility of isolated outer hair cells of guinea pig were studied in the experiments. Pretreatment with taicatoxin (0.19 microM) was able to prevent both the cell shortening induced by high K(+) (50mM), and the cell elongation induced by ionomycin (10 microM). These effects of taicatoxin can be mimicked by pretreatment of cells with Ca(2+)-free medium on the slow motility in response to ionomycin or high K(+). Pretreatment with neither calcium channel blockers such as nifedipine (L-type blocker), omega-conotoxin GVIA (N-type blocker), and omega-agatoxin IVA (P-type blocker); nor potassium channel blockers, such as tetraethylammonium chloride (TEA) and 3,4-diaminopyridine (3,4-DAP) can antagonize the cell shortening effect induced by high K(+) and cell elongation induced by ionomycin. The calcium-imaging experiment indicated that taicatoxin, but not nifedipine, did prevent an increase of intracellular Ca(2+) level significantly induced by high K(+). These results demonstrate that the effect of taicatoxin was to block the calcium entry through calcium channels of cell membrane, without relative to its properties of potassium channel blockers. We conclude that taicatoxin-sensitive-calcium channels at least impart, play a significant role in the slow motility of outer hair cell.

The effect of L-arginine on slow motility of mammalian outer hair cell.

The effect of L-arginine on the slow motility of mammalian cochlear outer hair cells was studied in this experiment. L-Arginine (3 mM) but not D-arginine (3 mM) or other amino acids (L-aspartate or L-glutamate) induced length increases of guinea pig outer hair cell. Similarly, the membrane-permeant cGMP analogues, 8-(4-chlorophenylthio)guanosine 3':5'-cyclic monophosphate (1 mM) or 8-bromo-guanosine 3':5'-cyclic monophosphate (1 mM) induced length increases of guinea pig outer hair cells. These length increases induced by L-arginine can be attenuated by a 30 min preincubation of the cells with the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride (3 mM) or 7-nitroindazole (1 mM). Comparing the effects of L-arginine and ionomycin on cell length and intracellular calcium change in outer hair cells, both L-arginine and ionomycin were able to induce the elongation of outer hair cells but L-arginine did not change the fluorescence intensity of Fluo-3. Preincubation of the cells with EGTA (3 mM) for 40 min to reduce the extracellular calcium concentration did not influence the effect of L-arginine. This experiment demonstrated that nitric oxide/cGMP pathway involvement in regulating the slow motility of mammalian outer hair cells cannot be ruled out. The effect of L-arginine is independent of extracellular calcium concentration.

Tissue distribution of different mercurial compounds analyzed by the improved FI-CVAAS.

Mercury contents in biological samples can be measured by cold vapor atomic absorption spectroscopy combined with the flow-injection analysis system. However, water vapor in the absorption cell attenuated and distorted the signals. This study described the strategy to overcome this problem by adding an additional gas-liquid separator after the mixing/separator assembly. This modification can efficiently minimize the moisture in the transfer line and in the absorption cell. This improved technique was adopted to study the differential tissue distribution of methylmercury and HgS after oral administration to mice for five consecutive days. The present study suggests that the insoluble HgS (the main constituent of a Chinese mineral drug, cinnabar, used as a sedative) can still be absorbed from gastrointestinal tract and distributed to various tissues including the brain. As compared with methylmercury, the total amount of HgS accumulated in the tissues ranging about one five-thousandth of methylmercury, which is well correlated with the biological activity of HgS reported previously.

Luteolin attenuates the pulmonary inflammatory response involves abilities of antioxidation and inhibition of MAPK and NFκB pathways in mice with endotoxin-induced acute lung injury.

Acute lung injury (ALI) in critically ill patients remains the leading cause of mortality and morbidity. Lipopolysaccharide (LPS) is a key mediator of lung injury. This study investigates the protective effects and mechanisms of luteolin in intratracheal instillation of LPS (100µg)-induced ALI in mice. Pretreatment of mice with 70µmol/kg luteolin significantly restores LPS-induced decrease in oxygen pressure and increase in carbon dioxide in arterial blood. The histopathological study established 70µmol/kg luteolin pretreatment markedly attenuates lung histopathological changes, such as haemorrhaging, interstitial edema, and infiltration of polymorphonuclear neutrophils (PMNs) into the lung parenchyma and alveolar spaces. Sufficient evidence for luteolin (35 and 70µmol/kg) suppresses activation and infiltration of PMNs is obtained in expression of surface marker CD11b and Ly6G on cells in bronchoalveolar lavage fluid (BALF) cells and myeloperoxidase activity in lung tissue. Furthermore, luteolin reduces the activity of catalase and superoxide dismutase, and the level of oxidative damage, and lipid peroxidation, in lung tissue. In addition, the secretion of TNF-α, KC, and ICAM-1 in the BALF after LPS challenge are also inhibited by luteolin. Moreover, luteolin reduced LPS-induced activation of MAPK and NFκB pathways. Therefore, luteolin is a potential protective antagonists for LPS-induced ALI in mice.

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils.

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O(2)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2µM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, ßI and ßII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca(2+)-dependent PKC in rat neutrophils.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils.

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O(2)(-)) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgammaS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40(phox), p47(phox) and p67(phox), phosphorylation of Ser residues in p47(phox), association between p47(phox) and p22(phox), and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47(phox) were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47(phox), moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca(2+) concentration ([Ca(2+)](i)) elevation in the presence but not in the absence of extracellular Ca(2+), and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia.

Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappaB (NF-kappaB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPbeta, but not those of p65, p50, C/EBPdelta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNgamma-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPbeta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPbeta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.