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Regulation of heme metabolism and cytochrome P-450 levels in primary culture of rat hepatocytes in a defined medium 总被引:1,自引:0,他引:1
Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme. 相似文献
3.
Randle LE Goldring CE Benson CA Metcalfe PN Kitteringham NR Park BK Williams DP 《Toxicology》2008,243(3):249-260
The Keap1-Nrf2-ARE signalling pathway has emerged as an important regulator of the mammalian defence system to enable detoxification and clearance of foreign chemicals. Recent studies by our group using paracetamol (APAP), diethylmaleate and buthionine sulphoximine have shown that for a given xenobiotic molecule, Nrf2 induction in the murine liver is associated with protein reactivity and glutathione depletion. Here, we have investigated, in vivo, whether the ability of four murine hepatotoxins, paracetamol, bromobenzene (BB), carbon tetrachloride (CCl4) and furosemide (FS) to deplete hepatic glutathione (GSH) is related to induction of hepatic Nrf2 nuclear translocation and Nrf2-dependent gene expression. Additionally, we studied whether hepatic Nrf2 nuclear translocation is a general response during the early stages of acute hepatic chemical stress in vivo. Male CD-1 mice were administered APAP (3.5 mmol/kg), FS (1.21 mmol/kg), BB (4.8 mmol/kg) and CCl4 (1 mmol/kg) for 1, 5 and 24h. Each compound elicited significant serum ALT increases after 24h (ALT U/L: APAP, 3036+/-1462; BB, 5308+/-2210; CCl4, 5089+/-1665; FS, 2301+/-1053), accompanied by centrilobular damage as assessed by histopathology. Treatment with APAP also elicited toxicity at a much earlier time point (5h) than the other hepatotoxins (ALT U/L: APAP, 1780+/-661; BB, 161+/-15; CCl4, 90+/-23; FS, 136+/-27). Significant GSH depletion was seen with APAP (9.6+/-1.7% of control levels) and BB (52.8+/-6.2% of control levels) 1h after administration, but not with FS and CCl4. Western Blot analysis revealed an increase in nuclear Nrf2, 1h after administration of BB (209+/-10% control), CCl4 (146+/-3% control) and FS (254+/-41% control), however this was significantly lower than the levels observed in the APAP-treated mice (462+/-36% control). The levels of Nrf2-dependent gene induction were also analysed by quantitative real-time PCR and Western blotting. Treatment with APAP for 1h caused a significant increase in the levels of haem oxygenase-1 (HO-1; 2.85-fold) and glutamate cysteine ligase (GCLC; 1.62-fold) mRNA. BB and FS did not affect the mRNA levels of either gene after 1h of treatment; however CCl4 significantly increased HO-1 mRNA at this time point. After 24h treatment with the hepatotoxins, there was evidence for the initiation of a late defence response. BB significantly increased both HO-1 and GCLC protein at this time point, CCl4 increased GCLC protein alone, although FS did not alter either of these proteins. In summary, we have demonstrated that the hepatotoxins BB, CCl4 and FS can induce a small but significant increase in Nrf2 accumulation in hepatic nuclei. However, this was associated with modest changes in hepatic GSH, a delayed development of toxicity and was insufficient to activate an early functional adaptive response to these hepatotoxins. 相似文献
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Jun-Yan Liu Bora Inceoglu Arzu Ulu Nipavan Chiamvimonvat 《Biochemical pharmacology》2010,79(6):880-887
Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects. 相似文献
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The present study was designed to investigate the effects of p-methoxyl-diphenyl diselenide (OMePhSe)2 on oxidative stress and renal damage parameters of mice exposed to cisplatin. (OMePhSe)2 (50 and 100 mg/kg/day) was orally administered to mice for six consecutive days. On the third day after the beginning of (OMePhSe)2 treatment, the renal toxicity was induced by injecting cisplatin (10 mg/kg intraperitoneal) in mice. (OMePhSe)2 treatment (50 mg/kg) partially reduced plasma urea and creatinine levels increased by cisplatin. Histopathological examination of kidneys showed that (OMePhSe)2 ameliorated renal injury caused by cisplatin. (OMePhSe)2 attenuated the decrease in reduced glutathione (GSH) and ascorbic acid (AA) levels, the inhibition of glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and catalase (CAT) activities caused by cisplatin in kidney. (OMePhSe)2 treatment partially protected against the inhibition of renal δ-aminolevulinic dehydratase (δ-ALA-D) activity caused by cisplatin. No alteration in renal lipid peroxidation levels was found in cisplatin and/or (OMePhSe)2 groups. (OMePhSe)2 was effective against the increase in reactive species (RS) levels caused by the cisplatin exposure. Based on the renoprotective and antioxidant actions of (OMePhSe)2 we suggest that this organoselenium compound could be considered a feasible candidate to protect against toxicity commonly encountered in cisplatin exposure. 相似文献
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Aldo-keto reductase (AKR) 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Delta(4)-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Delta(4)-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development. 相似文献
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Human Immunodeficiency Virus type 1 (HIV-1) integrase (IN) is an attractive target for the development of new antiviral therapies. Recently, several HIV-1 recombinant IN (rIN) in vitro inhibitors have been described. However, the great majority of them failed to block the virus replication in cell-based assays, suggesting the inadequacy of the in vitro assay systems used for inhibitor screening. To improve these systems, we designed a 40(mer) duplex DNA reaction substrate consisting of recognition sequences from both U3 and U5 HIV-1 long terminal repeat (LTR) termini. The HIV-1 rIN was able to catalyze its enzyme activities recognizing both ends of the 40(mer) dsDNA. Using this substrate we assayed the effects on rIN catalysis of different classes of compounds which inhibit the HIV-1 rIN in vitro when the reaction substrate is the standard 21(mer) U5 dsDNA, and that are either active or inactive on the HIV-1 replication. We also compared the efficacy of these compounds when added to the reaction before or after the formation of the rIN-dsDNA complex. In this system, the enzyme preincubation with the two-ended 40(mer) dsDNA before the addition of the compounds allowed a strong correlation between the effects of hydroxylated aromatics derivatives on rIN activity in cell-free assays and their effects on viral replication in cell-culture assays. This increase in drug selectivity of the rIN in vitro assay was explored by investigating whether it was due to the length of the 40(mer), longer than the standard 21(mer), or to presence of both viral ends, versus only one viral end. To this purpose we designed four 40(mer) oligonucleotides containing either only one viral end or two-repetitive ends, finding that the architecture of the rIN-dsDNA complex and its compound susceptibility is significantly influenced by the sequence of the dsDNA substrate. 相似文献
8.
Pinar Erkekoglu Walid Rachidi Belma Giray Mehmet Ozturk Filiz Hincal 《Toxicology and applied pharmacology》2010,248(1):52-62
Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates’ testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 μM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status. 相似文献
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Mirko Papritz Christine Pohl Michaela Moisch Maria Iris Hermanns Charles James Kirkpatrick 《Toxicology and applied pharmacology》2010,245(3):361-369
Cadmium (Cd2+) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier.In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell® filter plate. This coculture model was exposed to various concentrations of CdCl2.The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd2+ concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd2+ was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd2+ from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca2+ could prevent the barrier-disrupting effect of Cd2+.In conclusion, the exposure of Cd2+ to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd2+. 相似文献
11.
The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated. 相似文献
12.
Physiologically based biokinetic model of bioactivation and detoxification of the alkenylbenzene methyleugenol in rat 总被引:1,自引:0,他引:1
Ala′ A.A. Al-Subeihi Bert SpenkelinkNovalia Rachmawati Marelle G. BoersmaAns Punt Jacques VervoortPeter J. van Bladeren Ivonne M.C.M. Rietjens 《Toxicology in vitro》2011,25(1):267-285
The present study defines a physiologically based biokinetic (PBBK) model for the alkenylbenzene methyleugenol in rat based on in vitro metabolic parameters determined using relevant tissue fractions, in silico derived partition coefficients, and physiological parameters derived from the literature. The model was based on the model previously developed for the related alkenylbenzene estragole and consists of eight compartments including liver, lung, and kidney as metabolizing compartments, and separate compartments for fat, arterial blood, venous blood, richly perfused and slowly perfused tissues. Evaluation of the model was performed by comparing the PBBK predicted concentration of methyleugenol in the venous compartment to methyleugenol plasma levels reported in the literature, by comparing the PBBK predicted dose-dependent percentage of formation of 2-hydroxy-4,5-dimethoxyallylbenzene, 3-hydroxy-4-methoxyallylbenzene, and 1′-hydroxymethyleugenol glucuronide to the corresponding percentage of metabolites excreted in urine reported in the literature, which were demonstrated to be in the same order of magnitude. With the model obtained the relative extent of bioactivation and detoxification of methyleugenol at different oral doses was examined. At low doses, formation of 3-(3,4-dimethoxyphenyl)-2-propen-1-ol and methyleugenol-2′,3′-oxide leading to detoxification appear to be the major metabolic pathways, occurring in the liver. At high doses, the model reveals a relative increase in the formation of the proximate carcinogenic metabolite 1′-hydroxymethyleugenol, occurring in the liver. This relative increase in formation of 1′-hydroxymethyleugenol leads to a relative increase in formation of 1′-hydroxymethyleugenol glucuronide, 1′-oxomethyleugenol, and 1′-sulfooxymethyleugenol the latter being the ultimate carcinogenic metabolite of methyleugenol. These results indicate that the relative importance of different metabolic pathways of methyleugenol may vary in a dose-dependent way, leading to a relative increase in bioactiviation of methyleugenol at higher doses. 相似文献
13.
Girolami F Abbadessa G Racca S Spaccamiglio A Piccione F Dacasto M Carletti M Gardini G Di Carlo F Nebbia C 《Toxicology letters》2008,181(2):87-92
7,12-Dimethylbenzanthracene (DMBA) is an abundant environmental contaminant, which undergoes bioactivation, primarily by the CYP1 family, both in liver and extra-hepatic tissues. Dietary acetylsalicylic acid (ASA) has been recently reported to inhibit DMBA-mediated mammary tumour formation in rats. Chemopreventive substances may reduce the risk of developing cancer by decreasing metabolic enzymes responsible for generating reactive species (phase I enzymes) and/or increasing phase II enzymes that can deactivate radicals and electrophiles. To test these hypotheses, Sprague-Dawley female rats were orally administered ASA as lysine acetylsalicylate (50mg per capita/day for 21 days in water), DMBA (10mg per capita in olive oil on day 7, 14, and 21), ASA and DMBA in combination, and vehicles only, respectively. Six rats for each group were sacrificed on day 8, 15, and 22. The DMBA-mediated increase in hepatic CYP1A expression and related activities was not significantly affected by ASA, which, conversely, enhanced in a time-dependent manner the liver reduced glutathione content (up to 52%) and the activity of NAD(P)H-quinone oxidoreductase (up to 34%) in DMBA-treated rats. It is proposed that the positive modulation of the hepatic antioxidant systems by ASA may play a role in the chemoprevention of mammary tumourigenesis induced by DMBA in the female rat. 相似文献
14.
Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [3H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca2+]i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs. 相似文献
15.
Exploration of antioxidants of plant origin and their scientific validation for their immense pharmacological potential is emerging as an issue of intense research now-a-days.The effect of Cymbopogon citratus extract was seen on cell viability, oxidative stress markers i.e. ROS production, SOD activity, lipid peroxidation and GSH content of murine alveolar macrophages stressed with lipopolysaccharide. Modulation in release of NO and pro-inflammatory cytokine TNF-α along with alterations in mitochondrial membrane potential under stress were compared with known plant derived antioxidant quercetin. The extract was not found to be cytotoxic at any of the selected doses. At 5 and 10 μg the extract showed significant increase in SOD activity, GSH content (p < 0.001), decrease in ROS production as seen by fluorescent dye DCFH-DA and also MDA formation (lipid peroxidation marker) significantly. The extract also showed reduction in the release of pro-inflammatory mediators TNF-α and NO significantly indicating an anti-inflammatory effect. The extract was able to restore mitochondrial membrane potential as estimated by spectrofluorimetry using the fluorescent dye Rhodamine 123. The results suggest potential use of the cytoprotective, antioxidant and anti-inflammatory property of C. citratus in the form of dietary component and also in formulations against lung inflammatory diseases where oxidative stress plays an important role. 相似文献
16.
Min-Kyung Kim Huyn-Sun Lee Eun-Jin Kim Nam-Hee Won Young-Min Chi Byoung-Chul Kim Kwang-Won Lee 《Food and chemical toxicology》2007,45(9):1738-1744
The leaves of perilla [Perilla frutescens (L.) Britt. var. japonica (Hassk.) Hara] are often used in Asian gourmet food. The object of this study was to evaluate the protective effects of an aqueous extract of perilla leaves on the tert-butyl hydroperoxide (t-BHP)-induced oxidative injury observed in rat livers. The treatment of the hepatocytes with the perilla leaf extract (PLE) significantly reversed the t-BHP-induced cell cytotoxicity and lipid peroxidation. In addition, PLE exhibited ferric-reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activities. The in vivo study showed that the pretreatment with PLE (1000 or 3000 mg/kg) for 5 days before a single dose of t-BHP (i.p.; 0.2 mmol/kg) significantly lowered the serum levels of aspartate aminotransferase and alanine aminotransferase, reduced the indicators of oxidative stress in the liver, such as the glutathione disulfide content and lipid peroxidation level in a dose-dependent manner, and remarkably increased the activity of hepatic gamma-glutamylcysteine synthetase. Histopathological examination of the rat livers showed that PLE reduced the incidence of liver lesions induced by t-BHP. Based on the results described above, it is suggested that PLE has the potential to protect liver against t-BHP-induced hepatic damage in rats. 相似文献
17.
We have previously demonstrated that exogenous H2O2 stimulates Cl−/HCO3− exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H2O2-induced stimulation of Cl−/HCO3− exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H2O2, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H2O2 on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H2O2 had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H2O2 treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H2O2-induced stimulation of Cl−/HCO3− exchanger activity. It is concluded that H2O2-induced stimulation of Cl−/HCO3− exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H2O2, which contributes to their increased sensitivity to H2O2. 相似文献
18.
We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics of the cell lines in the FMP assay were in good agreement with previous findings in electrophysiology studies of the transporters. The FMP assay was capable of distinguishing between substrates and non-substrate inhibitors and to discriminate between "full" and "partial" substrates at the transporters. Taking advantage of the prolific nature of the FMP assay, interactions of the EAATs with substrates and inhibitors were studied in some detail. This is the first report of a high throughput screening assay for EAATs. We propose that the assay will be of great use in future studies of the transporters. Although conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands presently available is limited, and that almost all of these are characterized by low potency and a low degree of subtype selectivity, future screening of compound libraries at the EAAT-cell lines in the FMP assay could help identify structurally and pharmacologically novel ligands for the transporters. 相似文献
19.
Annalisa Bruno Luigia Di Francesco Isabella Coletta Maria Alessandra Alisi Claudio Milanese Emanuela Ricciotti Melania Dovizio Stefania Tacconelli Paola Patrignani 《Biochemical pharmacology》2010,79(7):974-981
Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH2 substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH2 metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE2, thromboxane (TX)B2, PGF2α and 6-keto-PGF1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE2 in human recombinant mPGES-1 with an IC50 of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE2 biosynthesis with an IC50 of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE2 biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB2, PGF2α and 6-keto-PGF1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE2 biosynthesis with an IC50 of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE2 generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH2 metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo. 相似文献
20.
Arsenic trioxide (ATO) is an effective therapeutic agent for the treatment of acute promyelocytic leukemia, but successful application of this agent may occasionally require the use of sensitizing strategies. The present work demonstrates that the flavonoids quercetin and chrysin cooperate with ATO to induce apoptosis in U937 promonocytes and other human leukemia cell lines (THP-1, HL-60). Co-treatment with ATO plus quercetin caused mitochondrial transmembrane potential dissipation, stimulated the mitochondrial apoptotic pathway, as indicated by cytochrome c and Omi/Htra2 release, XIAP and Bcl-X(L) down-regulation, and Bax activation, and caused caspase-8/Bid activation. Bcl-2 over-expression abrogated cytochrome c release and apoptosis, and also blocked caspase-8 activation. Quercetin and chrysin, alone or with ATO, decreased Akt phosphorylation as well as intracellular GSH content. GSH depletion was regulated at the level of L-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, and N-acetyl-L-cysteine failed both to restore GSH content and to prevent apoptosis. Treatment with BSO caused GSH depletion and potentiated ATO-provoked apoptosis, but did not affect apoptosis induction by ara-C and cisplatin. As an exception, ATO plus quercetin failed to elicit Akt de-phosphorylation and GSH depletion in NB4 acute promyelocytic leukemia cells, and correspondingly exhibited low cooperative effect in inducing apoptosis in this cell line. It is concluded that GSH depletion explains at least in part the selective potentiation of ATO toxicity by quercetin, and that this flavonoid might be used to increase the clinical efficacy of the antileukemic drug. 相似文献