An Avocado Constituent,Persenone A,Suppresses Expression of Inducible Forms of Nitric Oxide Synthase and Cyclooxygenase in Macrophages,and Hydrogen Peroxide Generation in Mouse Skin

We investigated the suppressive effects of an avocado constituent, persenone A, on lipopolysaccharide- and interferon-γ-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in a mouse macrophage cell line RAW 264.7. Persenone A at concentration of 20 μM almost completely suppressed both iNOS and COX-2 protein expression. In mouse skin, double treatments with persenone A (810 nmol) significantly suppressed double 12-O-tetradecanoylphorbol-13-acetate (TPA, 8.1 nmol) application-induced hydrogen peroxide (H₂O₂) generation. Treatment with persenone A before the second TPA treatment was sufficient to inhibit H₂O₂ generation, while the first treatment was not. This study thus suggests that persenone A is a possible agent to prevent inflammation-associated diseases including cancer.     

Arachidonic acid cascade inhibitors modulate phorbol ester-induced oxidative stress in female ICR mouse skin: differential roles of 5-lipoxygenase and cyclooxygenase-2 in leukocyte infiltration and activation

We recently developed a short-term assay for skin H2O2 generation induced by double 12-O-tetradecanoylphorbol-13-acetate (TPA) applications for mechanistic study on skin epidermal carcinogenesis. In the present study, we investigated the individual roles of arachidonic acid metabolism in H2O2 generation in mouse skin inflammation. The experiments using inhibitors of arachidonic acid (AA) metabolism showed that corticosteroid and a lipoxygenase (LO) inhibitor expectedly suppressed double TPA application-induced H2O2 generation through the interference of chemotactic action but not by direct decomposition or scavenging. We also demonstrated that the treatment of AA (1 mumol) and 5-LO metabolites including leukotriene B4 (LTB4) partly mimicked, though soybean LO-derived lipid hydroperoxide and prostaglandins did not, the priming effect evaluated by edema formation and leukocyte infiltration. We also confirmed that inflammatory leukocytes accumulated by LTB4 generated a significant amount of H2O2 by TPA stimulation. These results suggested that 5-LO metabolites of AA are the potential key molecules in the TPA-induced priming event. Interestingly, the cyclooxygenase (COX-) 2-selective inhibitor nimesulide (NS) and celecoxib (CXB) showed different responses than those of other inhibitors. These agents showed no specific potential to inhibit the priming event but significantly suppressed H2O2 generation, lipid peroxidation, and hyperplasia in mouse skin. From the results based on an in vitro leukocyte differentiation model, we speculated that the antioxidant effect of the COX-2 inhibitors might be partly associated with both counteraction of proinflammatory cytokine-enhanced ROS generation and inhibition of CD11b, an important molecule for cell adhesion, expression. Indeed, the topical application of NS attenuated the number of infiltrated leukocytes induced by TPA in mouse skin. Thus, these gathered data indicated the differential roles of 5-LO and COX-2 in leukocyte adhesion, infiltration, and H2O2 generation.     

Kurosu, a traditional vinegar produced from unpolished rice, suppresses lipid peroxidation in vitro and in mouse skin

The in vitro antioxidative activities of various kinds of vinegar were investigated by using a linoleic acid autoxidation model detected by the thiobarbituric acid (TBA) method and the 1,1-diphenyl-2-picrylhydrazyl radical system. An ethyl acetate extract of Kurosu (EK), a vinegar made from unpolished rice, exhibited the highest antioxidative activity in both systems. EK (5 mg) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema formation (14%) and myeloperoxidase activity (52%, P< 0.01) in female ICR mouse skin. Furthermore, EK significantly suppressed double TPA application-induced H2O2 generation (53%, P< 0.01) and lipid peroxidation determined by the TBA-reacting substance level (95 %, P< 0.01). In a two-stage carcinogenesis experiment with dimethylbenz[a]anthracene/TPA, EK significantly reduced the number of tumors per mouse by 36% (P<0.05) at 15 weeks after promotion. These results suggest that the antitumor-promoting effect may be partially due to the antioxidative properties of EK such as the decomposition of free radicals and interference with free radical-generating leukocytes.     

Inhibition by (+)-persenone [corrected] A-related compounds of nitric oxide and superoxide generation from inflammatory leukocytes

We have previously reported that persenone A, isolated from avocado fruit, is an effective inhibitor of both nitric oxide (NO) and superoxide (O2-) generation in cell culture systems. In this study, we have prepared four persenone A-related compounds and examined their inhibition of NO and O2- generation from inflammatory leukocytes. Some structural importance in persenone A to attenuate free radical generation is discussed.     

Pro/antioxidant Status in Murine Skin Following Topical Exposure to Cumene Hydroperoxide Throughout the Ontogeny of Skin Cancer

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.     

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.     

A JNK1/AP-1-dependent, COX-2 induction is implicated in 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation through regulating cell cycle progression

Cyclooxygenase-2 (COX-2) is reported to be one of the early-response gene products induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the relevance of COX-2 in TPA-induced cell transformation and the underlying mechanisms remains to be explored. Initially, we verified COX-2 induction after TPA treatment in mouse embryonic fibroblasts (MEF) and mouse epidermal cells Cl 41. More importantly, introduction of COX-2 small interfering RNA in MEFs or Cl 41 cells suppressed the cell transformation caused by TPA treatment. This inhibition could be reversed by overexpression of human full-length COX-2, indicating that COX-2 is at least one of the critical molecules involved in TPA-induced cell transformation. We further showed that TPA-promoted cell cycle progression was partially suppressed by COX-2 small interfering RNA, indicating that COX-2 also participated in TPA-associated cell cycle progression. Investigation of the upstream signaling pathways revealed that c-Jun-NH(2)-kinase 1 (JNK1), but not JNK2, played important roles in COX-2 induction, because knockout of JNK1 gene rather than JNK2 gene markedly impaired COX-2 induction. Furthermore, inhibition of c-Jun/activator protein 1 pathway or JNKs/c-Jun pathway by overexpression of dominant negative mutants of c-Jun, or MKK4 and MKK7 together, resulted in impairment of COX-2 induction, suggesting that JNK1/c-Jun/activator protein 1 pathway is involved in TPA-associated COX-2 induction. In contrast, IKK/p65 nuclear factor-kappaB pathway was not implicated because knockout of IKKalpha, IKKbeta, or p65 gene did not affect COX-2 induction although nuclear factor-kappaB was activated by TPA. In addition, the TPA-promoted cell cycle progression was found impaired in JNK1-deficient, but not in JNK2-deficient, MEFs. Our results show that JNK1-associated COX-2 induction is implicated in TPA-associated cell transformation and cell cycle progression.     

A sesquiterpenol extract potently suppresses inflammation in macrophages and mice skin and prevents chronic liver damage in mice through JNK-dependent HO-1 expression

This study aimed to elucidate the anti-inflammatory and hepatoprotective bioactivities of a sesquiterpenol, (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one (BSL), isolated from Cryptomeria japonica (Taxodiaceae) wood extract. BSL markedly suppressed TNF-α and IL-6 secretion, PGE(2) production, and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated mouse macrophages. BSL also potently inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced protein levels of nitrotyrosine and COX-2 in mouse skin with dermatitis. Conversely, the stress protein heme oxygenase-1 (HO-1) was found upregulated in the same BSL-treated macrophages, probably through activation of the JNK-dependent pathway. LPS-induced activation of NF-κB and mitogen-activated protein kinase signaling pathways, however, was not responsive to BSL treatment. A BSL-enriched extract (BSL-E; 10mg/kg) significantly prevented CCl(4)-induced chronic liver injury, lipid accumulation, and cell necrosis and inhibited aminotransferase activities and iNOS and COX-2 overexpression in mice liver tissues, an effect comparable with that of silymarin, a hepatoprotective drug.     

Hydrogen peroxide generation in whole rat pancreatic islets; synergistic regulation by cytoplasmic free calcium and protein kinase-C

This is the first report to show that pancreatic islet cells generate H2O2 and this H2O2 generation is regulated synergistically by cytoplasmic free calcium ([Ca2+]i) and protein kinase-C. Effects of calcium ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter, on H2O2 generation were studied in whole pancreatic islets obtained from male Wistar rats. We employed A23187 to elevate cytoplasmic free calcium, and TPA to activate protein kinase-C and monitored continuously their effects on H2O2 generation, measured using homovanillic acid and horseradish peroxidase. A23187 stimulates H2O2 generation. TPA, which activates protein kinase-C, augments this A23187-stimulated H2O2 generation. H2O2 generation is stimulated by an increase in [Ca2+]i and regulated synergistically by [Ca2+]i and protein kinase-C.     

Inhibition by tectorigenin and tectoridin of prostaglandin E2 production and cyclooxygenase-2 induction in rat peritoneal macrophages.

Tectorigenin and tectoridin, isolated from the rhizomes of Korean Belamcanda chinensis (Iridaceae) which are used as Chinese traditional medicine for the treatment of inflammation, suppressed prostaglandin E2 production by rat peritoneal macrophages stimulated by the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the endomembrane Ca2+-ATPase inhibitor, thapsigargin. Tectorigenin inhibited prostaglandin E2 production more potently than tectoridin. Neither compound inhibited the release of radioactivity from [3H]arachidonic acid-labeled macrophages stimulated by TPA or thapsigargin. In addition, activities of isolated cyclooxygenase (COX)-1 and COX-2 were not inhibited by the two compounds. Western blot analysis revealed that the induction of COX-2 by TPA or thapsigargin was inhibited by the two compounds in parallel with the inhibition of prostaglandin E2 production. These findings suggest that one of the mechanisms of the anti-inflammatory activities of the rhizomes of Belamcanda chinensis is the inhibition of prostaglandin E2 production by tectorigenin and tectoridin due to the inhibition of the induction of COX-2 in the inflammatory cells.     

Anti-inflammatory action of dietary fish oil and calorie restriction

Inflammation, inflammatory mediators, cyclooxygenase (COX)-2, and inducible nitric oxide (iNOS) are all influenced by age-related oxidative status. To investigate the effect of dietary fish oil (FO) and calorie restriction (CR) on oxidative stress-related inflammatory status with age, (NZB/NZW) F1 (B/W) mice were fed for 4 and 9 months either ad libitum or calorie-restricted (60% of ad libitum intake) diets containing 5% corn oil or 5% FO. We measured several key oxidative and inflammatory markers: TBARS, xanthine oxidase (XOD)-derived superoxide generation, and PGE2 and LTB4 production. Expressions of renal COX-1, COX-2, and iNOS mRNA were analyzed by RT-PCR; additionally, COX-2 protein was estimated by Western-blot method. Results show that FO intake and CR individually and together suppressed age-related increases in lipid peroxidation and superoxide generation. The inhibitory effects of dietary FO and CR were also found for iNOS expression, COX-2 expression, which subsequently led to the suppression of PGE2 and LTB4. We conclude that the beneficial effects of FO feeding and CR are synergistic in ameliorating the age-related nephritis of B/W mice by suppressing COX-2 and iNOS, reactive species generation, and pro-inflammatory mediators.     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Phorbol ester-mediated suppression of cytochrome P450 Cyp1a-1 induction in murine skin: involvement of protein kinase C.

Epidermal 7-ethoxyresorufin O-deethylase (EROD) activity was elevated greater than 100-fold within 4 to 7 h of topical treatment of SENCAR mice with 100 nmol dibenz[a,c]anthracene (DB[a,c]A). Treatment of skin with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) 2 to 8 h prior to DB[a,c]A application suppressed induction by 80%. Suppression was dose-dependent over the range of 0.01 to 5 micrograms TPA (ID50 approximately 0.6 nmol). EROD activities in normal and TPA-treated epidermis paralleled steady state P450 CYP1A1 mRNA content. Analogs of TPA incapable of activating or down-regulating protein kinase C (PKC) did not suppress induction. Pretreatment of skin with sn-1,2-didecanoylglycerol, an activator of PKC which causes translocation but no down-regulation, did not suppress EROD induction. However, induction was suppressed by chrysarobin, an anthralin analog that causes PKC down-regulation in the absence of prior activation. These studies suggest that PKC participates in the processes associated with Cyp1a-1 induction and that TPA effects Cyp1a-1 induction through its down-regulation of PKC.