Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

Saturated fatty acid exposure induces androgen overproduction in bovine adrenal cells.

BACKGROUND: Polycystic ovary syndrome (PCOS) is mainly defined by hyperandrogenemia, from ovarian and adrenal origin, and is characterized by insulin resistance (IR). Studies found that raising in vivo non-esterified fatty acid (NEFA) levels, which induces lipotoxicity, increases androgen levels and IR. The aim of this study was therefore to determine the effects of in vitro over-exposure to NEFA on androgen synthesis in a bovine adrenocortical cell model. METHODS: Bovine fasciculata/reticularis cells were cultured for 2days in the absence or presence of ACTH (10nmol/L) or Forskolin (fsk, 10µmol/L), alone or in combination with the saturated fatty acid (FA) palmitate (100µmol/L). Steroid production was measured in medium and corrected for initial cell seeding count. CYP17 protein expression and ERK1/2 phosphorylation were assessed by Western blotting. RESULTS: Under unstimulated conditions, dehydroepiandrosterone (DHEA) levels were barely detected and no difference was observed after palmitate exposure, which was also the case for CYP17 expression and ERK1/2 phosphorylation. Under stimulation, palmitate exposure increased DHEA production by 38% and 69%, for ACTH and fsk, respectively, as compared to untreated conditions (Ps⩽0.05). In palmitate-treated vs untreated cells, fsk-stimulated ERK1/2 phosphorylation was reduced by 46% (P=0.0047), but stimulated CYP17 expression was not significantly affected. CONCLUSION: In a model of androgen-producing cells, under stimulated conditions, overexposure to saturated FAs significantly increases androgen production and reduces MEK/ERK activation. Therefore, this study is the first to demonstrate that lipotoxicity can directly trigger androgen overproduction in vitro, in addition to its well-described impact on IR, which strongly supports a central role of lipotoxicity in PCOS pathophysiology.

Production of prostaglandins, isoprostanes and thromboxane by Aspergillus fumigatus: identification by gas chromatography-tandem mass spectrometry and quantification by enzyme immunoassay.

Aspergillus fumigatus has been reported to produce prostaglandin (PG)-like substances. The molecular structure of these fungal eicosanoids is however still unknown. By using the gas chromatography-tandem mass spectrometry (GC-MS/MS) methodology we identified a number of eicosanoids that were formed upon incubation of the precursor arachidonic acid ethyl ester (AAE, 10 µM) with three strains of A. fumigatus. The eicosanoids identified include the prostaglandins (PG) PGE(2), 6-keto-PGF(1α) (the stable hydrolysis product of prostacyclin PGI(2)) and PGF(2α), the isoprostanes 15(S)-8-iso-PGF(2α) and 15(S)-8-iso-PGE(2), and thromboxane B(2) (TxB(2), the stable hydrolysis product of TxA(2)). These eicosanoids are identical with those produced by cyclooxygenases (COX) in humans. The biosynthesis of all of these eicosanoids could not be inhibited by the human COX inhibitors indomethacin (100 µM), acetylsalicylic acid (100 µM) or the non-selective human lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (100 µM). By contrast, the selen-containing antioxidant ebselen (100 µM) was found to inhibit their synthesis. Our results suggest that mammals and fungi employ different eicosanoid biosynthesis pathways. As some of the detected eicosanoids are potent immunomodulators and bronchoconstrictors, they could play a possible role in pulmonary diseases associated with A. fumigatus infection.

Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression.

Although the literature suggests a protective (anabolic) effect of insulin-like growth factor-1 (IGF-1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF-1 signaling are advantageous for promoting an increase in life span through reduction in oxidative stress-induced tissue damage. To better understand this paradox, we utilized the hepatocyte-specific IGF-1 transgenic (HIT) mice, which exhibit 3-fold increases in serum IGF-1, with normal IGF-1 expression in other tissues, and mice with an IGF-1 null background that exclusively express IGF-1 in the liver, which thereby deliver IGF-1 by the endocrine route only (KO-HIT mice). We found that in the total absence of tissue igf1 gene expression (KO-HIT), increases in serum IGF-1 levels were associated with increased levels of lipid peroxidation products in serum and increased mortality rate at 18 months of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF-1 plays an important role in preserving trabecular bone architecture as KO-HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO-HIT mice, increases in serum IGF-1 levels were insufficient to protect against age-related muscle loss.

Resveratrol inhibits isoprostane production in young and aged rat brain.

Resveratrol (3,5,4-trihydroxystilbene), a viniferin polyphenolic compound, has been shown to have neuroprotective effects and we tested its possible antioxidant activity in young and aged rat brain, evaluating, in vitro, synaptosomal 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) production as a marker of oxidative stress. We found that in young rat brain synaptosomes resveratrol perfusion had no effect on basal 8-iso-PGF2alpha production, but quenched to basal levels the increased 8-iso-PGF2alpha production induced by hydrogen peroxide. On the other hand, in aged rats, resveratrol was able to decrease 8-iso-PGF2alpha production both basally and after hydrogen peroxide-induced oxidative stimulus. In conclusion, our findings show that the antioxidant effects of resveratrol in rat brain could play a neuroprotective role in aging, when the increased burden of oxidative stress is faced by defective antioxidant mechanisms.

Differential effects of N-acetylcysteine, theophylline or bicarbonate on contrast-induced rat renal vasoconstriction.

BACKGROUND: Vasoconstriction and reactive oxygen species (ROS) accumulation following contrast media (CM) injection are the key factors triggering CM-induced nephropathy. We compared the effects of N-acetylcysteine (NAC), theophylline or sodium bicarbonate on intrarenal vasoconstriction and ROS generation in a rat model of CM-induced nephropathy. METHODS: Following a 3-day dehydration, Sprague-Dawley rats received CM (Telebrix) or sham 'CM' injection of 0.9% saline. Part of them received NAC, theophylline or bicarbonate prior to CM. Medullar renal blood flow was estimated by laser Doppler. The animals were sacrificed 1, 15 or 30 min after the respective treatments, their kidneys allocated and intrarenal STAT-8 isoprostane, PGE(2) and NO assessed. RESULTS: Vasoconstriction was significantly attenuated by NAC. Theophylline only mildly attenuated the perfusion drop at 15 min, and was ineffective following 30 min. Unlike theophylline or bicarbonate, NAC significantly augmented intrarenal PGE(2). NAC, theophylline but not bicarbonate, gradually increased intrarenal NO. In all experimental variables, CM-induced ROS accumulation, represented by STAT-8 isoprostane estimation, progressed undisturbed. CONCLUSIONS: (1) CM-induced intrarenal vasoconstriction was efficiently prohibited by NAC but not bicarbonate or theophylline; (2) the vasodilatory effect of NAC was mediated via increased PGE(2) synthesis, and (3) ROS accumulation was a primary renal response to CM-induced injury, not affected by any pharmacologic manipulations.

Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy.

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

Formation of highly reactive cyclopentenone isoprostane compounds (A3/J3-isoprostanes) in vivo from eicosapentaenoic acid.

Omega-3 (omega-3) polyunsaturated fatty acids (PUFAs) found in marine fish oils are known to suppress inflammation associated with a wide variety of diseases. Eicosapentaenoic acid (EPA) is one of the most abundant omega-3 fatty acids in fish oil, but the mechanism(s) by which EPA exerts its beneficial effects is unknown. Recent studies, however, have demonstrated that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and anti-proliferative effects. Very few studies to date have investigated which EPA oxidation products are responsible for this bioactivity. Our research group has previously reported that anti-inflammatory prostaglandin A(2)-like and prostaglandin J(2)-like compounds, termed A(2)/J(2)-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether cyclopentenone-IsoP compounds are formed from the oxidation of EPA in vivo. Herein, we report the formation of cyclopentenone-IsoP molecules, termed A(3)/J(3)-IsoPs, formed in abundance in vitro and in vivo from EPA peroxidation. Chemical approaches coupled with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to structurally characterize these compounds as A(3)/J(3)-IsoPs. We found that levels of these molecules increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 +/- 0.4 ng/mg EPA to 196 +/- 23 ng/mg EPA after 36 h. We also detected these compounds in significant amounts in fresh liver tissue from EPA-fed rats at basal levels of 19 +/- 2 ng/g tissue. Amounts increased to 102 +/- 15 ng/g tissue in vivo in settings of oxidative stress. These studies have, for the first time, definitively characterized novel, highly reactive A/J-ring IsoP compounds that form in abundance from the oxidation of EPA in vivo.

Isoprostane formation and inhibition in atherothrombosis.

Enhanced oxidant stress plays a pivotal role in the pathogenesis of several conditions such as atherothrombosis, cancer and neurodegeneration. The availability of reliable assays of isoprostanes in biological fluids has prompted clinical investigations into the pathophysiological role of lipid peroxidation in cardiovascular disease. Established risk factors for coronary heart disease have been associated with enhanced oxidant stress, leading to increased lipid peroxidation and non-enzymatic formation of bioactive isoprostanes. These risk factors include diabetes mellitus, hypercholesterolemia, obesity and hyperhomocysteinemia. Clinical studies support the hypothesis that enhanced lipid peroxidation may contribute, at least in part, to persistent platelet activation.

N-acetylcysteine inhibits interleukin-17-induced interleukin-8 production from human airway smooth muscle cells: a possible role for anti-oxidative treatment in chronic lung rejection?

BACKGROUND: Long-term survival of lung transplantation is threatened by obliterative bronchiolitis, or its clinical equivalent, bronchiolitis obliterans syndrome. With a prevalence of >50% at 5 years after transplantation, it has emerged as the most significant long-term complication. Neutrophilic inflammation and increased interleukin (IL)-8 production seem to be part of the basic pathophysiologic mechanism of chronic rejection. Recently, it has been suggested that reactive oxygen species may also play an important role in the pathogenesis because they are known to induce smooth muscle proliferation. METHODS: Human airway smooth muscle cells in vitro were stimulated with IL-17 (0.1 to 10 ng/ml) or with IL-17 (10 ng/ml) and the anti-oxidative agent N-acetylcysteine (1 micromol/liter to 10 mmol/liter). Production of 8-isoprostane was measured with a commercially available enzyme immunoassay kit and production of IL-8 was measured using a standard enzyme-linked immunoassay technique. RESULTS: IL-17 produced a concentration-dependent increase in 8-isoprostane with a maximum of 136.5 +/- 15.5 pg/ml with IL-17 (10 ng/ml, p < 0.001, n = 12, vs unstimulated cells). N-acetylcysteine (NAC) was able to decrease IL-17-induced 8-isoprostane production, with a maximum decrease of 59.3 +/- 9% (p < 0.001, n = 12) with 10 mmol/liter of N-acetylcysteine, which also decreased IL-17-induced IL-8 production in a concentration-dependent manner (with maximum inhibition of 86.3% when combined with NAC 10 mmol/liter as compared with IL-17 alone). CONCLUSIONS: We demonstrated that human airway smooth muscle cells, when stimulated with IL-17, are able to produce 8-isoprostane, which could be inhibited by adding N-acetylcysteline, and which was also able to decrease IL-17-induced IL-8 production. The clinical significance of these in vitro findings for prevention or treatment of chronic rejection after lung transplantation remains to be investigated.

Ablation of the heat shock factor-1 increases susceptibility to hyperoxia-mediated cellular injury.

High concentrations of oxygen (hyperoxia) are known to cause cellular injury and death. The heat shock response is a highly conserved cellular defense mechanism that protects cells against various environmental stressors, including hyperoxia. Herein we determined the role of heat shock factor-1 (HSF-1), a major component of the heat shock response, in protecting cells against hyperoxia. Embryonic fibroblasts from HSF-1-null mutant mice (HSF-1 -/- cells) were compared to wild-type embryonic fibroblasts (HSF-1 +/+ cells) following 24 hours' exposure to room air or hyperoxia (95% O(2)). Acute survival in hyperoxia was decreased in HSF-1 -/- cells as compared to HSF-1 +/+ cells. Intracellular ATP levels were significantly lower in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Isoprostane levels, a marker of membrane lipid peroxidation, were significantly higher in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Restoration of HSF-1 in the HSF-1 -/- cells by stable transfection with a HSF-1 expression plasmid improved survival in hyperoxia when compared to HSF-1 -/- cells stably transfected with the empty expression vector. Hyperoxia increased activation of HSF-1 in HSF-1 +/+ cells and in HSF-1 -/- cells stably transfected with the HSF-1 expression plasmid. These data demonstrate that HSF-1 plays an important role in conferring resistance to hyperoxia in vitro.

Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function.

Oxidative stress can impair proteasome function, both of which are features of neurodegenerative diseases. Inhibition of proteasome function leads to protein accumulation and cell death. We discovered recently the formation of highly reactive g-ketoaldehydes, isoketals (IsoKs), and neuroketals (NeuroKs) as products of the isoprostane and neuroprostane pathways of free radical-induced lipid peroxidation that are analogous to cyclooxygenase-derived levuglandins (LGs). Because aldehydes that are much less reactive than IsoKs have been shown to inhibit proteasome function, we explored the ability of the proteasome to degrade IsoK-adducted proteins/peptides and the effect of IsoK and IsoK-adducted proteins/peptides on proteasome function. Adduction of IsoK to model proteasome substrates significantly reduced their rate of degradation by the 20S proteasome. The ability of IsoK to inhibit proteasome function directly was observed only at very high concentrations. However, at much lower concentrations, an IsoK-adducted protein (ovalbumin) and peptide (Ab1-40) significantly inhibited chymotrypsin-like activity of the 20S proteasome. Moreover, incubation of IsoK with P19 neuroglial cultures dose-dependently inhibited proteasome activity (IC50 = 330 nM) and induced cell death (LC50 = 670 nM). These findings suggest that IsoKs/NeuroKs/LGs can inhibit proteasome activity and, if overproduced, may have relevance to the pathogenesis of neurodegenerative diseases.