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Biomedical subjects

Pierre Leroy

Publications and source records attributed to Pierre Leroy.

8 recordsLinked to original sources
PubMed2003

Different mechanisms for gamma-glutamyltransferase-dependent resistance to carboplatin and cisplatin.

In this work, we investigated the effect of gamma-glutamyltransferase (GGT) overexpression on cell viability after carboplatin treatment and compared with cisplatin. Carboplatin challenge of HeLa cells induced GGT and glutamate-cystine ligase (GCL) activities by 2- and 1.4-fold, respectively and concomitantly increased the intracellular reduced glutathione (GSH) level (1.5-fold). To study the role of GGT, HeLa-GGT cells, a stably transfected cell line overexpressing GGT (120-150 mU/mg protein) and the parental HeLa cells (10-15 mU/mg protein) were used. Both cell lines exhibited comparable viability (IC(50) approximately 150 microM) after carboplatin treatment when cultured in standard (250 microM cystine) medium. Culture in low (50 microM) cystine medium resulted in a dramatic decrease (approximately 90%) of the intracellular GSH level and to a 2.5-fold increase of carboplatin cytotoxicity (IC(50) approximately 60 microM). When GSH (50 microM) was included in the culture medium, only HeLa-GGT cells exhibited increased resistance to carboplatin. Using partially purified GGT from HeLa-GGT cells, we show that cisplatin forms adducts with cysteinylglycine, depending only on GGT activity whereas carboplatin did not efficiently react with cysteinylglycine. Thus, in this model system, GGT activity can affect platinum drugs cytotoxocity by two different ways: cisplatin can be detoxified extracellularly after reaction with the -SH group of cysteinylglycine; in the case of carboplatin, the supply of GSH precursors, initiated by GGT, increases the intracellular level of the tripeptide and provides enhanced defensive mechanisms to the cell.

Antineoplastic Agents↗
PubMed2003

Changes in serum retinol, alpha-tocopherol, vitamin C, carotenoids, xinc and selenium after micronutrient supplementation during alcohol rehabilitation.

OBJECTIVE: To test the effect of a 21-day supplementation with moderate doses of antioxidant nutrients on biochemical indicators of vitamin, carotenoid and trace element levels in alcohol-dependent patients during a program of alcohol rehabilitation. DESIGN: A randomized double-blind trial was performed comparing two groups receiving daily either a combination of micronutrients (beta-carotene: 6 mg, vitamin C: 120 mg, vitamin E: 30 mg, zinc: 20 mg, selenium: 100 micro g) or a placebo. SUBJECTS: 106 alcohol-dependent patients 20 to 60 years of age without severe liver disease, hospitalized for a 21-day rehabilitation program. Measure of Outcome: Vitamin C, retinol, alpha-tocopherol, zeaxanthin/lutein, beta-cryptoxanthin, lycopene, alpha- and beta-carotene, zinc and selenium were measured in serum, initially and after supplementation. RESULTS: (1) In the placebo group, after 21 days of rehabilitation, serum concentrations of vitamin C and all five carotenoids significantly increased, whereas retinol and alpha-tocopherol concentrations decreased; zinc and selenium levels were unaffected. (2) At the end of the hospital stay, serum indicators were significantly improved in the supplement group as compared to the placebo group for vitamin C, alpha-tocopherol, beta-carotene, zinc and selenium; conversely, lycopene changes were higher in the placebo group than in supplement group. (3) Of the serum antioxidants measured at entrance, only vitamin C was significantly depleted in heavy smokers, and, after the supplementation period, vitamin C was efficiently repleted in this later group. CONCLUSION: Our results indicate that a short-term supplementation with physiological doses of antioxidant vitamins, carotenoids and trace elements during alcohol rehabilitation clearly improves micronutrient status indicators. Heavy smokers in particular seem to respond to vitamin C supplementation.

Adult↗
PubMed2002

Enhanced resistance of HeLa cells to cisplatin by overexpression of gamma-glutamyltransferase.

Gamma-glutamyltransferase (GGT), which is a key enzyme for the cellular glutathione (GSH) homeostasis, was shown to be overexpressed in human tumor cells selected for resistance to cisplatin and to influence the resistance of experimental tumors in vivo. We first established that cisplatin treatment of HeLa cells was accompanied by an early 3-fold induction of GGT synthesis, enhancing the possibility that this enzyme plays an important role in the cell defenses against this anticancer drug. This role was then studied using a GGT-transfected HeLa cell line (HeLa-GGT) exhibiting 10 times the activity of the parental HeLa cells (120-150 and 10-14 mU/mg protein, respectively). Both cell lines showed comparable intracellular GSH levels and cisplatin resistance when cultured in high (250 microM) or low (50 microM) cysteine-containing medium. When 50 microM of GSH were included in the low-cysteine culture medium only HeLa-GGT cells partially recovered their intracellular GSH and exhibited an increased resistance to cisplatin. Cisplatin treatment also inhibited GGT-dependent production of reactive oxygen species, a process depending on the availability of cysteinylglycine produced during GSH catabolism. Furthermore, we showed that cisplatin forms adducts with cysteinylglycine 10 times more rapidly than with GSH, and that these adducts were formed only in the extracellular medium of HeLa GGT cells. This extracellular mechanism could at least partially account for the increased resistance of GGT-rich cells to cisplatin.

Antineoplastic Agents↗
PubMed2002

A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells.

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.

3T3 Cells↗
PubMed2002

gamma-Glutamyltranspeptidase-dependent metabolism of 4-hydroxynonenal-glutathione conjugate.

A major pathway for detoxification of the highly reactive lipid peroxidation product, 4-hydroxy-2,3-trans-nonenal (HNE) is through the conjugation with glutathione (GSH). We have studied the metabolism of GS-HNE conjugate by the enzyme gamma-glutamyltranspeptidase (GGT) using its purified form, as well as a GGT-overexpressing fibroblast cell line (V79 GGT). Using mass spectrometry analysis we identified for the first time cysteinylglycine-HNE (CysGly-HNE) as the GGT metabolite of GS-HNE. Furthermore, the GGT-dependent metabolism of GS-HNE in the V79 GGT cell line was associated with a considerable increase of cytotoxicity as compared to a control cell line which does not express GGT (V79 Cl). The cytotoxic effect was dose- and time-dependent (100% cellular death at 200 microM GS-HNE after 24 h incubation) in V79 GGT cells, whereas no decrease of viability was observed in V79 Cl cells. A similar cytotoxic effect was obtained when cells were incubated directly with CysGly-HNE, demonstrating that this GGT-dependent metabolite unlike GS-HNE, exhibits cytotoxic properties.

Aldehydes↗
PubMed2002

Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPH-dependent microsomal oxidative reactions: free radical scavenging ability of isoniazid.

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 >> acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.

Animals↗
PubMed2002

An isocratic liquid chromatographic method with diode-array detection for the simultaneous determination of alpha-tocopherol, retinol, and five carotenoids in human serum.

An isocratic high-performance liquid chromatography (HPLC) method for the simultaneous determination of alpha-tocopherol, retinol, and five carotenoids (lutein-zeaxanthin, beta-cryptoxanthin, lycopene, and alpha- and beta-carotene) in human serum is described. Serum samples are deproteinized with ethanol and extracted once with n-hexane. Resulting extracts are injected onto a C18 reversed-phase column eluted with methanol-acetonitrile-tetrahydrofuran (75:20:5, v/v/v), and full elution of all the analytes is realized isocratically within 20 min. The detection is operated using three channels of a diode-array spectrophotometer at 290, 325, and 450 nm for tocopherol, retinol, and the carotenoids, respectively. An internal standard is used for each channel, which improves precision. The choice of internal standards is discussed, as well as the extraction protocol and the need for adding an antioxidant during the extraction and chromatographic steps. The analytical recoveries for liposoluble vitamins and carotenoids are more than 85%. Intra-assay relative standard deviation (RSD) values (n = 20) for measured concentrations in serum range from 3.3% (retinol) to 9.5% (lycopene), and interassay RSDs (n = 5) range from 3.8% (alpha-tocopherol) to 13.7% (beta-cryptoxanthin). The present method is used to quantitate the cited vitamins in healthy subjects (n = 168) from ages 9 to 55 years old.

Adolescent↗