SYNOVIOCYTES BUT NOT ARTICULAR CHONDROCYTES RELEASE FREE RADICALS AFTER REPETITIVE CYCLES OF ANOXIA/REOXYGENATION: AN ELECTRON SPIN RESONANCE STUDY.

Poster (2005)

Objective : We investigated if short recurrent periods of anoxia/reoxygenation (A/R), mimicking the in vivo conditions of hypoxia, could stimulate the production of free radicals by synoviocytes and ... [more ▼]

Objective : We investigated if short recurrent periods of anoxia/reoxygenation (A/R), mimicking the in vivo conditions of hypoxia, could stimulate the production of free radicals by synoviocytes and chondrocytes. Material and Methods : Synoviocytes (immortalised rabbit line HIG-82 and equine synoviocytes isolated from synovial membranes of the stifle joint) and chondrocytes (isolated from the same equine joint) were cultured for 48 hours. The respiration rate of the cells (107 cells/assay) was studied by oxymetry and the free radical production monitored by electron spin resonance (ESR) in the presence of the spin trap (-[-4-pyridyl 1-oxide]-N-tert butyl nitrone)/ethanol mixture (2%v/v). The A/R consisted of three periods of 20 minutes anoxia, each anoxia period being followed by re-oxygenation. Oxygen consumption by the cells was measured before starting the anoxia/reoxygenation cycles and after each reoxygenation. At the end of the last A/R period, the cells were transferred into the ESR flat cell and in the cavity, and the free radical formation was monitored. Results : The equine chondrocytes showed a low respiration rate, weakly affected by A/R and no production of free radicals (n=3). Synoviocytes showed a higher respiration rate (at least 20 times higher) which was affected by recurrent A/R (decrease of the slope of oxygen consumption), and a free radical production as evidenced by the appearance of the 4-POBN/EtOH adducts (n=6). Conclusions :These observations suggest that synoviocytes, but not chondrocytes, responded to repeated anoxia-reoxygenation conditions by the production of free radicals. Synoviocytes could thus be responsible for the production of free radicals in the joints, what could be an important factor in the onset of osteoarthritis. [less ▲]

Quantification of lipid bilayer viscosity and fuidity effect induced by propofol.

Conference (2004, April 23)

Effects of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells.

in Biochemical and Biophysical Research Communications (2004), 318(4), 941-8

We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae ... [more ▼]

We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae [Biochem. Biophys. Res. Commun. 287 (2001) 781]. We describe here the modifications of the responses of Chlamydia-primed THP-1 cells to hydrocortisone (HCT) and methylprednisolone (MPL). HCT and MPL inhibited the production of the cytokines TNFalpha and IL-8. But HCT, which inhibited the respiratory burst in LPS-primed monocytes, paradoxically stimulated the phenomenon in Chlamydia-primed cells; MPL exerted no significant effect. Both glucocorticoids did not significantly modify the triggering effect of Chlamydia on NF-kappaB binding activity. On the expression of p22(phox), a protein subunit of the NADPH oxidase, HCT had an increasing and MPL a decreasing effect. Glucocorticoids thus had unexpected effects on the inflammatory response of Chlamydia-primed monocytes. [less ▲]

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)

in Biochemical and Biophysical Research Communications (2004), 325

Investigation of singlet oxygen reactivity towards propofol

in Photochemical & Photobiological Sciences (2003), 2(9), 939-945

The reaction between the anaesthetic agent 2,6-diisopropylphenol (propofol, PPF) and singlet oxygen (1O2) has been investigated in aqueous solution by means of HPLC, GC, absorption spectroscopy and laser ... [more ▼]

The reaction between the anaesthetic agent 2,6-diisopropylphenol (propofol, PPF) and singlet oxygen (1O2) has been investigated in aqueous solution by means of HPLC, GC, absorption spectroscopy and laser flash photolysis with infrared luminescence detection. The rate constants for the physical and chemical quenching of 1O2 by PPF (kPPF) are found to be 2.66 x 10(5) M(-1) s(-1) and approximately 3.2 x 10(6) M(-1) s(-1) in CD3OD and D2O-CD3OD (75:25 v/v), respectively. The reaction of propofol with singlet oxygen produced by light irradiation of Rose Bengal leads essentially to two reaction products, 2,6-diisopropyl-p-benzoquinone and 3,5,3',5'-tetraisopropyl-(4,4')-diphenoquinone that are unambiguously identified from comparison with authentic samples. [less ▲]

EFFECTS OF O2 TENSION AND GLUCOSE CONCENTRATION ON THE CELLULAR RESPIRATION OF EQUINE ARTICULAR CHONDROCYTES IN CULTURE.

Poster (2003)

In vivo, articular chondrocytes are exposed to 5 to 10% O2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation. We were interested to study the ... [more ▼]

In vivo, articular chondrocytes are exposed to 5 to 10% O2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation. We were interested to study the effects of O2 and glucose variations on cellular respiration, glucose consumption and lactate production. Equine articular chondrocytes were cultured in suspension for 2 days under 5 or 21 % O2 in the gaseous phase, and with 0, 1.0 or 4.5 g/L glucose. The viable cells were then counted and the respiration rate (O2 consumption) of 10.106 cells was monitored by oxymetry for 2 hours; after oxymetry, glucose and lactate were measured in the medium (enzymatic assays). After 2 days, the cell viability was the best at 5% O2 and 1g/L glucose; it decreased at 4.5 g/L glucose and was the worst at 0g/L glucose, for the two O2 tensions (n=3). There was no obvious difference of the respiration rate between cells cultured at 5 and 21% O2, but respiration of chondrocytes was surprisingly low. When cells were submitted to 20 min anoxia at 0% O2, the O2 consumption was doubled at re-oxygenation for cells previously cultured at 21% O2. Glucose and lactate values found in the medium after oxymetry: lactate release in medium was similar (36.23 and 34.57 mg/L respectively) for cells cultured with 1g glucose and 5 or 21% O2 conditions; lactate values were low (2.03 and 8,63 mg/L respectively) for 4.5 g glucose and 5 or 21% O2. Glucose uptake was not different whatever the culture conditions. These results indicate a low cellular respiration with a lactate production linked to the glucose concentrationin the medium, and raise the question of the capacity of chondrocytes to produce ROS in vivo starting from the mitochondrial chain. [less ▲]

Oxidant activity of rabbit synoviocytes (HIG-82) demonstrated by oxymetry and ethylene production.

Poster (2003)

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen ... [more ▼]

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen tensions and the oxidant activity of these cells in response to stimuli. Synoviocytes were cultured at 5 and 21 % O2, their O2 consumption (cellular respiration, monitored with Clark electrode) was measured at 21% O2 and after anoxia, before and after stimulation with phorbol myristate acetate (PMA), and their oxidant response to PMA stimulation was quantified by measuring ethylene (gas chromatography) released when the substrate, alpha-keto-gamma-methylbutyric acid, is oxidised by the ROS produced by the cells. Cell growth was faster at 21 % O2 than at 5% O2, and microscopic observation revealed 2 cell populations: a few small round cells in suspension and many adherent cells. By oxymetry, we observed that a 106 synoviocytes suspension in 2 ml completely consumed O2 within 15 min, that anoxia (7 min) slightly slowed the respiration rate down and that PMA stimulation increased O2 consumption (150 % increase). The oxidant activity (ethylene production) of the cells was stimulated by PMA in a dose-dependent manner (10-9 to 10-7M) but the cell response was highly variable (from 150 to 1500 % increase) and was largely reduced by diphenyliodonium, an inhibitor of NADPH-oxidase and NO-synthase. The capacity to produce free radical species was confirmed for the small round cells by detection of an electron paramagnetic resonance (EPR) signal after stimulation. These results thus demonstrate a sensibility to O2 and an oxidant activity of synoviocytes at least related to ROS production by NADPH-oxidase activity. [less ▲]

Investigation of the reaction of peroxynitrite with propofol at acid pH: Predominant production of oxidized, nitrated, and halogenated derivatives

in Nitric Oxide (2003), 8(3), 170-181

Oxidative Processes in Human Promonocytic Cells (Thp-1) after Differentiation into Macrophages by Incubation with Chlamydia Pneumoniae Extracts

in Biochemical and Biophysical Research Communications (2001), 287(3), 781-8

Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1 ... [more ▼]

Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1), the cells were differentiated into macrophages by preincubation with C. pneumoniae extract, and further stimulated by phorbol myristate acetate. In these conditions, the differentiated cells oxidized a thiol compound and released superoxide anion as demonstrated respectively by gas liquid chromatography and electron spin resonance. The thiol oxidation and superoxide anion release were inhibited by diphenyliodonium, a NADPH oxidase and NOsynthase inhibitor, proving that the respiratory burst and the NOsynthase were involved in the oxidation processes occurring in the differentiated THP-1. The role of H(2)O(2) (derived from superoxide anion) was indicated by the enhancing effect of a peroxidase on the thiol oxidation. The presence of alpha-tocopherol in the surrounding medium strongly diminished the oxidation of the thiol target. [less ▲]

Electrooxidation Potential as a Tool in the Early Screening for New Safer Clozapine-Like Analogues

in Journal of Medicinal Chemistry (2001), 44(5), 769-76

The chemical modification of clozapine (1) has permitted the finding of new analogues, e.g., olanzapine (2), quetiapine (3), 5-(4-methylpiperazin-1-yl)-8-chloropyrido[2,3-b][1,5]benzoxazepine fumarate (9 ... [more ▼]

The chemical modification of clozapine (1) has permitted the finding of new analogues, e.g., olanzapine (2), quetiapine (3), 5-(4-methylpiperazin-1-yl)-8-chloropyrido[2,3-b][1,5]benzoxazepine fumarate (9), with a clinical or psychopharmacological profile similar to that of clozapine. However, when developing new derivatives, the designers are discouraged by the development of clozapine-induced agranulocytosis. Different researchers have raised the role played by the oxidizability of the molecule in such a deleterious effect. In the present paper, we examined the oxidation profile (direct scavenging abilities, efficacy in inhibiting lipid peroxidation, and electrooxidation potential) of newly developed methoxy and trifluoromethylsulfonyloxy analogues related to clozapine, some of them being described as putative antipsychotic. The oxazepine derivative 7, unlike the other diazepine derivatives (6, 10--12), was not readily oxidized. Using a statistical predictive model for hematotoxicity previously described, 7 was found in the cluster of potentially nontoxic compounds while diazepine derivatives 6 and 10-12 were classified as potentially toxic compounds. Among these original compounds, 7, which presents a preclinical clozapine-like profile and a low sensitivity to oxidation, could be a promising antipsychotic candidate with low side effects. Considering the tricyclic derivatives examined so far, some elements of structure-oxidation relationship (SOR) might be pointed out. Regarding the nature of the tricyclic ring substituent, from the most to the least sensitive to oxidation, the sequence was as follows: HO > Cl > CH(3)O > CF(3)SO(2)O. The nature of the tricyclic ring influenced also the sensitivity to oxidation; the diazepine moiety appeared to be the most reactive ring compared to oxa- and thiazepine congeners. These parameters could be advantageously integrated in the early design of new safer clozapine-like analogues. [less ▲]