Giovanna Santini

Effects of Vitamin E Supplementation on F2-Isoprostane and Thromboxane Biosynthesis in Healthy Cigarette Smokers

BACKGROUND Increased formation of 8-iso-prostaglandin (PG) F(2alpha) and thromboxane (TX) A(2), potent agonists of platelet and vascular thromboxane (TH)/PGH(2) receptors, has been detected in cigarette smokers. We performed a randomized, double-blind, placebo-controlled study of the effects of vitamin E (300, 600, and 1200 mg/d, each dose for 3 consecutive weeks) on 8-iso-PGF(2alpha) and TXA(2) biosynthesis in 46 moderate cigarette smokers. METHODS AND RESULTS Urinary immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2), plasma vitamin E, and serum TXB(2) were measured by previously validated techniques. Baseline urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion averaged 241+/-78 and 430+/-293 pg/mg creatinine, respectively. Urinary 8-iso-PGF(2alpha) was significantly correlated with 11-dehydro-TXB(2) (r=0.360, n=138, P<0.0001). Baseline plasma vitamin E levels averaged 20.6+/-4.9 micromol/L and were inversely correlated with urinary 11-dehydro-TXB(2) (r=-0.304, P=0.039) but not with 8-iso-PGF(2alpha) (r=-0.227, P=0.129). Vitamin E supplementation caused a dose-dependent increase in its plasma levels that reached a plateau at 600 mg (42.3+/-11.2 micromol/L, P<0. 001). This was not associated with any statistically significant change in urinary 8-iso-PGF(2alpha) or 11-dehydro-TXB(2) excretion. CONCLUSIONS Supplementation with pharmacological doses of vitamin E has no detectable effects on lipid peroxidation and thromboxane biosynthesis in vivo in healthy subjects with a mild degree of oxidant stress. These findings are consistent with the hypothesis that the basal rate of lipid peroxidation is a major determinant of the response to vitamin E supplementation and have implications for the use of vitamin E in healthy subjects as well as for the design and interpretation of clinical trials of antioxidant intervention.

Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indanone (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases

1 We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti‐inflammatory drugs, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]methanesulphonamide (NS‐398) and 5‐methanesulphonamido‐6‐(2, 4‐difluorothiophenyl)‐1‐indanone (L‐745, 337), in inhibiting the cyclo‐oxygenase activity of prostaglandin endoperoxide synthase‐2 (PGHS‐2) vs PGHS‐1 in human blood monocytes and platelets, respectively. 2 Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS‐1 and incubated at 37°C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml−1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo‐oxygenase activity of LPS‐induced monocyte PGHS‐2. 3 The effects of the same inhibitors on platelet PGHS‐1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin‐free periods, to clot at 37°C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4 Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo‐oxygenase activity of both PGHS‐1 and PGHS‐2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L‐745, 337 and NS‐398 achieved selective inhibition of monocyte PGHS‐2 (IC50 ratio: > 150). L‐745, 337 and NS‐398 did not affect LPS‐induced monocyte PGHS‐2 biosynthesis to any detectable extent. 5 We conclude that L‐745, 337 and NS‐398 are selective inhibitors of the cyclo‐oxygenase activity of human monocyte PGHS‐2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.

Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation

1 The isoprostane 8‐epi‐prostaglandin (PG)F2α is produced by free radical‐catalyzed peroxidation of arachidonic acid. It may also be formed as a minor product of the cyclo‐oxygenase activity of platelet PGH synthase (PGHS)‐1. We investigated 8‐epi‐PGF2α production associated with induction of the human monocyte PGHS‐2 and its pharmacological modulation. 2 Heparinized whole blood samples were drawn from healthy volunteers, 48 h following oral dosing with aspirin 300 mg to suppress platelet cyclo‐oxygenase activity. One ml aliquots were incubated with lipopolysaccharide (LPS: 0.1–50 μg ml−1) for 0–24 h at 37°C. PGE2 and 8‐epi‐PGF2α were measured in separated plasma by radioimmunoassay and enzyme immunoassay techniques. 3 Levels of both eicosanoids were undetectable (i.e. < 60 pg ml−1) at time 0. LPS induced the formation of PGE2 and 8‐epi‐PGF2α in a time‐ and concentration‐dependent fashion, coincident with the induction of PGHS‐2 detected by Western blot analysis of monocyte lysates. After 24 h at 10 μg ml−1 LPS, immunoreactive PGE2 and 8‐epi‐PGF2α averaged 10,480 ± 4,643 and 295 ± 140 pg ml−1 (mean ± s.d., n = 6), respectively. 4 Dexamethasone and 5‐methanesulphonamido‐6‐(2, 4‐difluorothiophenyl)‐1‐indanone (L‐745,337), a selective inhibitor of the cyclo‐oxygenase activity of PGHS‐2, reduced PGE2 and 8‐epi‐PGF2α production in response to LPS. 5 Isolated monocytes produced PGE2 and 8‐epi‐PGF2α in response to LPS (10 μg ml−1) in a time‐dependent fashion. Monocyte PGE2 and 8‐epi‐PGF2α production was largely prevented by dexamethasone (2 μm) and cycloheximide (10 μg ml−1) in association with suppression of PGHS‐2 but not of PGHS‐1 expression. 6 We conclude that the induction of PGHS‐2 in human monocytes is associated with cyclo‐oxygenase‐dependent generation of the vasoconstrictor and platelet‐agonist 8‐epi‐PGF2α.

Effects of nimesulide on constitutive and inducible prostanoid biosynthesis in human beings

The aim of this study was to test the hypothesis that nimesulide, a nonsteroidal antiinflammatory drug, or its principal metabolite 4‐hydroxynimesulide, is a selective inhibitor of prostaglandin H synthase‐2 in human beings.

Characterization of Chemical Inhibitors of Brefeldin A-activated Mono-ADP-ribosylation

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 ± 3.1 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing theV max of the reaction, whereas it does not influence the K m of the enzyme for NAD+(154 ± 13 μm). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

The human pharmacology of monocyte cyclooxygenase 2 inhibition by cortisol and synthetic glucocorticoids

We studied the concentration dependence of the inhibitory effects of cortisol, 6‐methylprednisolone, and dexamethasone on cyclooxygenase‐2 (COX‐2) expression and activity in human monocytes in response to lipopolysaccharide (LPS) in vitro. Moreover, we characterized the time and dose dependence of the inhibitory effects of 6‐methylprednisolone, administered to healthy subjects, on LPS‐inducible prostaglandin E2 (PGE2) biosynthesis in whole blood ex vivo.

Effects of flurbiprofen and flurbinitroxybutylester on prostaglandin endoperoxide synthases.

The aim of our study was to evaluate the selectivity of flurbiprofen and flurbinitroxybutylester for inhibition of the cyclooxygenase activity of prostaglandin endoperoxide synthase-2 vs. prostaglandin endoperoxide synthase-1 in human blood monocytes and platelets, respectively. In whole blood, flurbiprofen was approximately 10-fold more potent that flurbinitroxybutylester to inhibit the cyclooxygenase activity of platelet prostaglandin endoperoxide synthase-1 (IC50 microM: 0.90 +/- 0.27 vs. 10.70 +/- 5, mean +/- S.D., P < 0.05). In contrast, the 2 compounds were equipotent to inhibit prostaglandin endoperoxide synthase-2 cyclooxygenase activity in whole blood (IC50 microM: 0.90 +/- 0.25 vs. 0.80 +/- 0.35) or isolated monocytes (IC50 microM: 0.03 +/- 0.02). Neither flurbiprofen nor flubinitroxybutylester (0.28-112 microM) affected prostaglandin endoperoxide synthase isozyme expression by lypopolysaccharide-stimulated monocytes. In whole blood, flurbinitroxybutylester was slowly converted to flubiprofen and this in turn could influence the extent of inhibition of the cyclooxygenase activity of prostaglandin endoperoxide synthase-1. In conclusion, the addition of a nitroxybutyl moiety to flurbiprofen seems to reduce its capacity to inhibit the cyclooxygenase activity of prostaglandin endoperoxide synthase-1. Whether this effect will result in a reduced risk of gastrointestinal toxicity remains to be studied in man.

Cyclooxygenase-Independent Induction of P2LWAF-1/CIP1, Apoptosis and Differentiation by L-745, 337 and Salicylate in HT-29 Colon Cancer Cells

A large body of experimental evidence indicates that in animal models of bowel cancer, nonsteroidal antiinflammatory drugs (NSAIDs) can suppress carcinogenesis1,2. Moreover, both case-control and cohort studies have consistently found that regular use of aspirin is associated with approximately 50% reduction in the incidence and mortality of colorectal cancer, both in men and women1,3. Thirdly, sulindac reduces the number and size of colorectal adenomas in patients with familial adenomatous polyposis (FAP), though its effect is incomplete and reversible1,2.

Characterization of the Endogenous Mono-ADP-Ribosylation Stimulated by Brefeldin A

We have recently described a novel enzymatic mono-ADP-ribosyl transfer reaction induced by brefeldin A, a well characterized inhibitor of vesicular traffic, which selectively modifies two cytosolic proteins of 38 kDa (p38) and 50 kDa (BARS-50). p38 was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme and a multifunctional protein involved in several cellular processes; BARS-50 might be a novel G protein, since it is able to bind GTP and the beta gamma subunit of G proteins. We have characterized this enzymatic activity and screened in vitro the effects of different drugs belonging to the coumarine (dicumarol, coumermicin A1 and novobiocin) and quinone (ilimaquinones, benzoquinones and naphtoquinones) class. These drugs blocked the BFA-dependent mono-ADP-ribosylation, showed remarkable effects on Golgi morphology in control cells, and antagonized the tubular reticular redistribution of the Golgi complex in brefeldin A treated cells (see papers of Corda and Colanzi in this issue) suggesting a possible role for ADP-ribosylation in both the cellular effects of brefeldin A and the maintenance of the structure/function of the Golgi complex.

COX-2 expression and inhibition in human monocytes

The conversion of arachidonic acid to prostaglandin (PG) H2 is catalysed by PG-endoperoxide synthase (PGHS) which exhibits both cyclooxygenase (COX) and peroxidase activities1. PGH2 is further metabolized by other enzymes to various prostanoids (PGs, prostacyclin and thromboxane A2). Two isozymes of PGHS exist, referred to as PGHS-1 and PGHS-2 or COX-1 and COX-22. COX-1 is a constitutive enzyme present in almost all cell types3, and is the major isoform found in gastrointestinal tissue4. Prostanoid production by COX-1 is involved in physiological functions such as vascular homeostasis, control of kidney function and gastric cytoprotection2. COX-2 is induced in a more restricted, cell-specific fashion by mitogenic and inflammatory stimuli5–12.