Superoxide radical production by allopurinol and xanthine oxidase

Oxypurinol, an inhibitor of xanthine oxidase (XO), is being studied to block XO-catalyzed superoxide radical formation and thereby treat and protect failing heart tissue. Allopurinol, a prodrug that is converted to oxypurinol by xanthine oxidase, is also being studied for similar purposes. Because allopurinol, itself, may be generating superoxide radicals, we currently studied the reaction of allopurinol with xanthine oxidase and confirmed that allopurinol does produce superoxide radicals during its conversion to oxypurinol. At pH 6.8 and 25 degrees C in the presence of 0.02 U/ml of XO, 10 and 20 microM allopurinol both produced 10 microM oxypurinol and 2.8 microM superoxide radical (determined by cytochrome C reduction). The 10 microM allopurinol was completely converted to oxypurinol, while the 20 microM allopurinol required a second addition of xanthine oxidase to complete the conversion. Fourteen percent of the reducing equivalents donated from allopurinol or xanthine reacted with oxygen to form superoxide radicals. Superoxide dismutase prevented the reduction of cytochrome C by these substrates. At higher xanthine oxidase concentrations, or at lower temperatures, more of the 20 microM allopurinol was converted to oxypurinol during the initial reaction. At lower xanthine oxidase concentrations, or higher temperatures, less conversion occurred. At pH 7.8, the amount of superoxide radicals produced from allopurinol and xanthine was nearly doubled. These results indicate that allopurinol is a conventional substrate that generates superoxide radicals during its oxidation by xanthine oxidase. Oxypurinol did not produce superoxide radicals.     

Degradation of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone in U937 cells

We examined whether any changes were induced in cellular proteins by an inhibitor of acylpeptide hydrolase (ACPH) (EC 3.4.19.1), acetylleucine chloromethyl ketone (ALCK), which was shown in our previous report to induce apoptosis of human U937 cells. Extract prepared from U937 cells in 0.05% Triton X-100-PBS was incubated with ALCK at 37 degrees, and then analyzed using SDS-PAGE. A 36kDa protein in the cell extract was decreased markedly during the incubation period. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) by its specific enzyme activity, N-terminal amino acid sequence, and Western blotting. Incubation of purified GAPDH with ALCK resulted in a decrease of GAPDH activity, but not in a decrease in the amount of GAPDH. The ALCK-induced GAPDH decrease in the cell extract was abrogated by co-incubation with a serine protease inhibitor, diisopropyl fluorophosphate, suggesting that GAPDH was first inactivated by ALCK, and subsequently degraded by a serine protease(s). GAPDH degradation was also observed in U937 cell cultures in the presence of ALCK. The significance of GAPDH inhibition in the apoptotic process is discussed.     

Deregulation of glycolysis in cancer: glyceraldehyde-3-phosphate dehydrogenase as a therapeutic target

Introduction: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme, but recent studies have shown its non-glycolytic role in cell death, survival mechanisms and diseases. Increase in glycolysis, in particular overexpression of GAPDH, has been considered an important feature of many types of cancer cells. This review focuses on the role of GAPDH in carcinogenesis and the possibility of using this target for anticancer therapy.

Areas covered: In this review, the studies targeting GAPDH in human cancer as well as its functions in normal and cancer cells are described and discussed.

Expert opinion: GAPDH is an essential component of the glycolysis energy system, which is actively employed in cancer cells. Analysis of the so-called bioenergetics signature (the ratio of beta-F1-ATPase and GAPDH proteins) of different cancer types can be used for estimation of the cell metabolic activity, cancer aggressiveness and response to chemotherapy. Recent studies suggest GAPDH as a promising target for therapy of some carcinomas. Incidentally, limitations of this approach may come from the versatility of the GAPDH enzyme, since it combines glycolytic, pro-apoptotic and other activities. Hence, targeting GAPDH may lead to unexpected results concerning normal cells and therefore requires further research.     

Structural basis for the thermal stability of glyceraldehyde-3-phosphate dehydrogenases

The amino acid sequences of two thermophilic and five mesophilic glyceralde-hyde-3-phosphate dehydrogenases have been compared with the known three-dimensional structure of this enzyme to determine the factors responsible for thermal stability. The changes are greatest in the S-loop regions at the center of the tetramer, which show a quantitative increase in hydrophobicity and polarity that can strengthen subunit interactions in a complementary manner. The S-loops also show increases in residue volume and bulk that may indicate a tighter packing at the molecular center. In addition, there are changes in the secondary structural parameters indicating that the helices, in particular, may be more stable in the thermophilic proteins. Increases in the hydrophobicity of domain and subunit contacts for the Thermus aquaticus glyceraldehyde-3-phosphate dehydrogenase may explain why it is the most thermostable protein in this series.     

槲皮素对在体大鼠心肌缺血再灌性心律失常的保护作用

本文用在体Wistar大鼠心肌缺血再灌注模型,观察槲皮素对缺血再灌性心律失常的保护作用。在再灌前1min至再灌后2min静脉滴注槲皮素(0.5mmol/L,10ml/kg),可显著缩短心律失常的持续时间,降低室颤的发生率、再灌注区心肌组织中MDA的含量及XOD的活性,而对SOD具有明显的保护作用。结果提示槲皮素的抗心肌缺血再灌性心律失常作用可能与抑制心肌组织中OFR的形成和保护心肌组织中SOD或直接清除OFR有关。     

Differential agonist-induced regulation of human M2 and M3 muscarinic receptors

We have compared the regulation of M(2) and M(3) muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration-dependently reduced M(2) receptor density with a maximum reduction of about 60%. Treatment with 1mM carbachol for 24hr was accompanied by desensitisation of carbachol-induced Ca(2+) elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein alpha-subunits. Consistently, heterologous desensitisation of Ca(2+) elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time- and concentration-dependently up-regulated M(3) receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M(3) receptor was a reduction in the expression of the alpha-subunit of G(q/11). The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca(2+) elevation. However, the reduction of G(q/11) was accompanied by a heterologous desensitisation of Ca(2+) elevations by ATP and sphingosine-1-phosphate. Levels of M(2) and M(3) receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M(2) and M(3) receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.     

Effector-induced dissociation of glyceraldehyde-3-phosphate dehydrogenase discriminated by urea solvation

The dissociation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) from pig muscle in water solutions (0.1 M phosphate, pH 7) at increased urea concentrations was studied by means of frontal-gel chromatography, intrinsic (TRP) fluorescence, differential absorption spectroscopy and selective chemical modification at TRP-193. The results are in agreement with a consecutive two-step model of dissociation of the tetramer and the dimer (C*_T= 0.42 M urea < C*_D= 1.39 M urea). The binding of effector(s) destabilizes the oligomeric structures (ΔG^T changes from -1.00 to -0.54 kcal/mol; ΔG^D from -2.30 to -1.22 kcal/mol). The introduction of the bulky Koshland-reagent group to TRP-193 at the subunit-subunit interface leads to a decrease of the stability with δΔG ? 1 kcal/mol, owing to TRP-193…TYR-39…TYR-92 cluster destruction. By using lobster GAPD atomic coordinates (PDB file 1GPD) and pig muscle GAPD amino-acid sequence, a tentative molecular model was constructed and the subunit contacts in terms of the Lee-Richard static accessibilities were described. A detailed analysis of the dissociation as a transfer of the buried residues from the molecular interface to the urea solutions was performed.     

3-磷酸甘油醛脱氢酶测定在糖尿病肾病诊断中的临床意义

目的通过测定血中3-磷酸甘油醛脱氢酶的活性,探讨GAPD在糖尿病肾病早期诊断中的意义。方法测定190例健康者和80例2型糖尿病患者的血GAPD,尿微量白蛋白和β2-微球蛋白,对结果进行统计分析。结果疾病组中GAPD活性水平均高于正常对照组,差异有统计学意义(P<0.05)。疾病组中的微量白蛋白尿组、大量白蛋白尿组中的微量白蛋白和β2-微球蛋白高于正常对照组,差异有统计学意义(P<0.05)。但是正常白蛋白尿组中的微量白蛋白和β2-微球蛋白与对照组相比差异无统计学意(P>0.05)。结论 GAPD在正常白蛋白尿期活性已经发生变化,比微量白蛋白和β2-微球蛋白活性变化更早,能够提早预测糖尿病肾脏疾病的发生,有利于临床做出诊断,改善患者的预后。     

Effect of an inactivator of glyceraldehyde-3-phosphate dehydrogenase, a fortuitous arsenate reductase, on disposition of arsenate in rats.

The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)-alpha-cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 micromol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.     

Further observations on in vitro and in vivo effects of 2,5-hexanedione on glyceraldehyde-3-phosphate dehydrogenase

The in vitro and in vivo effect of aliphatic diketones has been studied on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) d,l-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating EC 1.2.1.12 activity). Neurotoxic diketone, 2,5-hexanedione (2,5-HD), but not 2,4-hexanedione (2,4-HD), a non-neurotoxic diketone, inhibited GAPDH in rat brain homogenate preincubated with 25 mM diketones for 20 min. If the preincubation period was increased to 2 h, approximately 25% and 55% inhibition of GAPDH activity was observed with 1 mM and 5 mM 2,5-HD respectively. The inhibiton of GAPDH activity was also seen in sciatic nerves but not in the brain or liver homogenates of rats chronically intoxicated with 2,5-HD for 12 weeks. The inhibition of GAPDH by 2,5-HD appears to be selective, and thus confirms earlier data from this laboratory.     

alpha-Chlorohydrin inhibits glyceraldehyde-3-phosphate dehydrogenase in multiple organs as well as in sperm.

Numerous studies have documented inhibitory effects of alpha-chlorohydrin (ACH) on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in spermatozoa. A sperm-specific G3PDH isoform has been described. The possibility that ACH may inhibit G3PDH in cell types other than sperm was investigated in this work. In addition, the onset of ACH-induced epididymal toxicity was described. Changes to epididymal histology occurred 6 h following a single dose of ACH (50 mg/kg po) and were confined to the proximal initial segment. By 24 h, no epithelial cells lined the basement membrane of that region. Three h after ACH administration (50 mg/kg po), G3PDH activity was significantly decreased in sperm (85%) as well as in kidney (31%), liver (49%), and epididymis (35%). Enzyme activity remained inhibited at 6 and 24 h. G3PDH was immunolocalized in the epididymis and staining was highest in the efferent ducts and initial segment as well as in smooth muscle. Since G3PDH is a microtubule-associated protein and microtubule-dependent endocytosis occurs in the epididymis, beta-tubulin was also immunolocalized. beta-tubulin densely stained the apical region of initial segment and caput epithelial cells. Disruption of beta-tubulin immunostaining correlated with the localization and onset of the lesion. Co-localization of G3PDH and beta-tubulin immunostaining was not observed although both antibodies most densely stained the initial segment. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition. Moreover, ACH inhibition of G3PDH is not confined to sperm, although the sperm enzyme is most sensitive to inhibition.     

Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes

Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation which are directly involved in the CsA hepatotoxicity. As antioxidant, Vitamin E (VitE) has been used to diminish the toxicity of CsA in vitro. Besides its direct action as the classical antioxidant implicated in preventing lipid peroxidation, we decided to investigate the effect of VitE on the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD) catalase and glutathione peroxidase (GPx) on CsA cytotoxicity in primary cultures of rat hepatocytes. In cells incubated in the presence of CsA, there was an increase in the expression and activity of MnSOD and CuZnSOD but not in that of catalase and GPx. However, when hepatocytes were coincubated with CsA and VitE, an increase in the expression and activity in all antioxidant enzymes (MnSOD, CuZnSOD, catalase and GPx) was observed. In conclusion, we suggest (a) that the imbalance between SOD and catalase/GPx by the effect of CsA is the main mechanism responsible for peroxide accumulation and cell death in hepatocytes, and (b) that the presence of VitE in culture media reduces the oxidative stress through the inhibition of lipid peroxidation, but also through the increase of the expression and activity of catalase and GPx which allows the restoration of SOD and catalase/GPx coordination, indispensable for the correct cell defense against ROS.     

人参皂苷Rg3差异调节前列腺癌细胞PC3和DU145增殖

目的 探讨人参皂苷Rg3对前列腺癌细胞PC3和DU145细胞增殖的调节作用。方法 100 µmol/L人参皂苷 Rg3处理体外培养的前列腺癌细胞 PC3和 DU145,使用 CCK-8实验检测细胞的增殖能力;DCFH-DA活性氧荧光探针试剂盒检测人参皂苷 Rg3处理的 DU145细胞中活性氧（ROS）水平;JC-1法检测 PC3和 DU145细胞线粒体膜电位的去极化;RT-PCR和免疫印迹法检测PC3和DU145细胞中氧化还原相关蛋白的表达差异。结果 CCK-8实验结果表明,100 µmol/L人参皂苷 Rg3能够显著抑制 PC3细胞增殖,而对 DU145细胞的增殖没有显著调节作用;DCFHDA活性氧荧光探针试剂盒检测结果表明,人参皂苷 Rg3能够显著上调 DU145细胞和 PC3细胞中 ROS水平;JC-1检测实验表明,人参皂苷Rg3能够诱导PC3和DU145细胞中线粒体膜电位去极化;RT-PCR和免疫印迹实验表明,抗氧化蛋白谷胱甘肽过氧化物酶-1（GPX-1）和超氧化物歧化酶2（SOD2）在DU145细胞中的表达显著高于PC3细胞。结论 人参皂苷Rg3对PC3和DU145细胞的增殖表现出不同的调节作用,可能与细胞中抗氧化蛋白的表达差异有关。     

Spirulina or dandelion-enriched diet of mothers alleviates lead-induced damages in brain and cerebellum of newborn rats

This study was aimed at evaluating the toxic effects of a prenatal exposure to lead acetate on brain tissues of newborn rats, and potent protective effects of spirulina (Arthropira platensis) or dandelion (Taraxacum officinalis) added to rat diet. Female rats were given a normal diet (control) or a diet enriched with spirulina or dandelion. Additionally, lead acetate was administered to one half of these rats through drinking water from the 5th day of gestation, to day 14 postpartum. Lead toxicity was assessed by measuring blood lead levels, brain weight, tissue damage, as well as protein content, lipid peroxidation and activities of antioxidant enzymes in brain tissues of neonates. Lead poisoning of mothers caused lead deposition in the brain and cerebellum of newborns and cerebellum tissue damages. Moreover, a significant decrease in weight and protein content of these tissues was found. Oxidative stress and changes in antioxidant enzyme activities in brain tissues were also recorded. Conversely, no such damages or biochemical changes were found in neonates from plant fed lead-poisoned mothers. These results strongly suggest that beneficial effects of spirulina- or dandelion-added diet on lead-intoxicated rats proceeded through the reduction of the lead-induced oxidative stress and related damages.     

Beneficial effect of shallot (Allium ascalonicum L.) extract on cyclosporine nephrotoxicity in rats.

The clinical use of an immunosuppressive cyclosporine A (CsA) is limited by its serious nephrotoxic effect. Evidences have suggested the role of oxidative stress in its pathogenesis. Shallot (Allium ascalonicum L.) has recently been shown to possess antioxidative and free radical scavenging abilities. The present study was undertaken to investigate the possible beneficial effect of shallot extract on renal injury caused by CsA. Male Wistar rats were treated orally with vehicle, CsA (25 mg/kg), shallot extract (1 g/kg), and CsA plus shallot extract for 21 days. Renal function, histopathology, tissue malondialdehyde (MDA) and glutathione (GSH) levels were evaluated 24 h after the last treatment. CsA-induced nephrotoxicity was evidenced by increased blood urea nitrogen and serum creatinine, but decreased urea and creatinine clearance. The kidney of CsA treated rats exhibited severe vacuolations and tubular necrosis. CsA also induced oxidative stress, as indicated by increased renal MDA and reduced GSH concentrations. Administration of shallot extract along with CsA counteracted the deleterious effects of CsA on renal dysfunction, oxidative stress markers, and morphological changes. These data indicate the protective potential of shallot extract against CsA nephrotoxicity and suggest a significant contribution of its antioxidant property to this beneficial effect.     

Preventive effect of neutropenia on carbon tetrachloride-induced hepatotoxicity in rats

The preventive effect of neutropenia on carbon tetrachloride (CCl4)-induced hepatotoxicity was examined in rats. In rats treated once with CCl4 (1 ml kg(-1), i.p.), the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indices of liver cell damage, and the hepatic activity of myeloperoxidase (MPO), an index of tissue neutrophil infiltration, increased at 6 h after the intoxication and further increased at 24 h. The liver of CCl4 -treated rats showed an increase in the concentration of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, and decreases in superoxide dismutase (SOD) activity and reduced glutathione (GSH) concentration at 6 h after the intoxication followed by a further increase in TBARS concentration and further decreases in SOD activity and GSH concentration at 24 h with increased xanthine oxidase (XO) activity at 24 h. Neutropenic treatment with anti-rat neutrophil antiserum (2 ml kg(-1), i.p.) at 0.5 h after CCl4 intoxication attenuated the increases in serum ALT and AST activities and hepatic MPO activity and TBARS concentration and the decreases in hepatic SOD activity and GSH concentration found at 6 and 24 h after CCl4 intoxication and the increase in hepatic XO activity found at 24 h after the intoxication. This neutropenia reduced the necrotic and degenerative changes with inflammatory cell infiltration in the liver cell of CCl4 -treated rats. These results indicate that neutropenia prevents CCl4 -induced hepatotoxicity in rats by attenuating the disruption of hepatic reactive oxygen species metabolism mediated by neutrophils accumulating in the liver tissue.     

Attenuation of cyclosporine A toxicity by sublethal heat shock. Role of catalase

Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases because of its specific inhibiting effect on signal transduction pathways of cell T receptor. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation, which are directly involved in the CsA hepatotoxicity. In the present study, we investigated the effect of a sublethal heat pre-treatment (43 degrees C for 30 min) on the hepatoma cell line HepG2 exposed to cytotoxic concentrations of CsA (10 and 25 microM) for 3 and 24 h. Parameters of cytotoxicity were assayed by measuring LDH (lactate dehydrogenase) leakage into the medium. Peroxide concentration was tested by flow cytometry by measuring the fluorescence intensity of DCF (dichlorofluorescein). Gene expression of catalase was detected by measuring the respective mRNA and proteins, as well as protein level of HSP70. The enzymatic activity of catalase was also determined. Heat pre-treatment significantly reduced CsA cytotoxicity as well as the level of peroxide generation. The protective effect of the previous heat treatment (corroborated by the irreversible catalase inhibitor 3-aminotriazole) against the CsA cytotoxicity was due to an increased expression and activity of catalase that was significantly reduced by the effect of CsA. We conclude that heat pre-treatment strongly protects against CsA injury, and the mechanism of this protection is by means of inducing not only the expression of HSP70 but also the expression and activity of catalase, the main enzyme system involved in H(2)O(2) elimination.     

A prolyl oligopeptidase inhibitor, Z-Pro-Prolinal, inhibits glyceraldehyde-3-phosphate dehydrogenase translocation and production of reactive oxygen species in CV1-P cells exposed to 6-hydroxydopamine

We studied the ability of prolyl oligopeptidase (POP) inhibitors, Z-Pro-Prolinal and JTP-4819, to prevent translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and formation of reactive oxygen species (ROS), in 6-hydroxydopamine (6-OHDA) and cytosine arabinoside (Ara-C) treated monkey fibroblast (CV1-P) and human neuroblastoma (SH-SY5Y) cells. The cells were pretreated with POP inhibitors (30 min) before addition of toxicants. GAPDH was analyzed by Western hybridization, ROS by fluorescent 2′7′-dichlorodihydro-fluorescein diacetate, and viability by the MTT method. Both toxicants induced GAPDH translocation to the particulate fraction (mitochondria and nuclei). Z-Pro-Prolinal was able to inhibit the translocation in 6-OHDA-exposed CV1-P cells. In SH-SY5Y cells and in JTP-4819 pretreated cells, no prevention of translocation was seen. However, the intensity of GAPDH in cytosolic fraction increased. Both inhibitors blocked 6-OHDA-induced ROS-production to the control level in CV1-P but, not in SH-SY5Y cells, without affecting their viability. In conclusion, POP inhibitors are able to prevent certain cell stress related factors such as ROS production or GAPDH translocation.     

Styrene induces an inflammatory response in human lung epithelial cells via oxidative stress and NF-kappaB activation

Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-κB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-κB activity. An inhibitor of NF-κB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-κB signalling pathway by styrene is mediated via a redox-sensitive mechanism.     

Antioxidant effects of flavonoid from Croatian Cystus incanus L. rich bee pollen

Oxidant/antioxidant status, estrogenic/anti-estrogenic activity and gene expression profile were studied in mice fed with Cystus incanus L. (Cistaceae) reach bee pollen from location in Central Croatia’s Dalmatia coast and offshore islands. Seven phenolic compounds (out of 13 tested) in bee pollen sample were detected by high performance liquid chromatography (HPLC) analysis. Phenolics detected in C. incanus L. bee pollen belong to flavonol (pinocembrin), flavanols (quercetin, kaempferol, galangin, and isorhamnetin), flavones (chrysin) and phenylpropanoids (caffeic acid). Bee pollen as a food supplement (100 mg/kg bw mixed with commercial food pellets) compared to control (commercial food pellets) modulated antioxidant enzymes (AOE) in the mice liver, brain and lysate of erythrocytes and reduced hepatic lipid peroxidation (LPO). Bee pollen induced 25% of anti-estrogenic properties while no estrogenic activity was found. Differential gene expression profile analyses after bee pollen enriched diet identify underexpressed gene Hspa9a, Tnfsf6 (liver) and down-regulated gene expression of Casp 1 and Cc121c (brain) which are important in the apoptosis pathway and chemotaxis. These results indicate that used bee pollen possess a noticable source of compounds with health protective potential and antioxidant activity.