François Trivin

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.

Quantitative analysis of C60 fullerene in blood and tissues by high-performance liquid chromatography with photodiode-array and mass spectrometric detection

A high-performance liquid chromatographic (HPLC) assay method for C60 fullerene, in blood, liver and spleen using photodiode-array detection or mass spectrometric detection (LC-MS) and C70 fullerene, as the internal standard, is described. The recovery from mouse blood and tissues spiked with micronized C60 exceeds 90%. The method is linear from 0.05 to 200 mg of C60 per liter of blood and from 0.05 to 5.00% of C60 per tissue weight. The limit of detection of the method is 0.1 ng of C60 per injection. This method was applied to mouse blood and tissue samples after intraperitoneal administration of a micronized C60 suspension.

Cytotoxicity of bilirubin for human fibroblasts and rat astrocytes in culture. Effect of the ratio of bilirubin to serum albumin

The sensitivity of rat brain astrocytes and human fibroblasts in culture to unconjugated bilirubin was investigated. Medium containing 6 mumol/1 bilirubin and increasing concentrations of human serum albumin giving ratios of 0.5-1.5 that resulted in an increase of the free bilirubin concentrations. The LDH activity in the culture medium was an index of cytolysis and the MTT assay was used as an index of mitochondrial impairment. The ratios producing half-maximum cell lysis after 24, 48, and 72 h, were 1.1, 0.9 and 0.85, for astrocytes, and 1.2, 0.75 and 0.75, for fibroblasts. Mitochondrial activity decreased after 24 h for ratio = 0.7 and partly recovered at 48 h. Mitochondrial activity was more impaired in fibroblasts than in astrocytes above ratio = 0.7. The cytotoxic effects were linked to the free bilirubin concentration. We conclude that astrocytes are less sensitive to bilirubin cytotoxic effects than are fibroblasts.

Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

Background: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. Methods: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. Results: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P=0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86±8.63 μmol/L versus 11.90 ± 3.25 μmol/L, P<0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. Conclusions: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

Early effects of C60 Administration in Swiss Mice: A Preliminary Account for In Vivo C60 Toxicity.

Abstract High amounts of micronized C60 have been injected intraperitoneally into Swiss mice. Until the fourteenth day, they were still alive without any behaviour trouble. C60 was well absorbed, and found localized in spleen and liver. Inside the liver, C60 was

High-performance liquid chromatographic determination of tauro- and glyco-conjugated bile acids in human serum

A method for the identification and individual determination of the ten tauro- and glyco-conjugated bile acids is described. It consists in a specific three-step extraction from small serum samples (500 microliters), high-performance liquid chromatographic separation and direct spectrophotometric detection at 119 nm. Extraction can be checked by the use of an internal standard. The reproducibility, recovery and separation of fractions were satisfactory.

The Influence of C60 Powders On Cultured Human Leukocytes

Abstract In order to check its possible acute toxicity, C60 was incorporated into living human phagocytes. It was observed that C60 has no influence on the survival of human leukocytes.

Hyperhomocysteinaemia and parameters of antioxidative defence in Tunisian patients with coronary heart disease

Abstract Background An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of this study was to test the parameters of antioxidative defence and to assess their association with hyperhomocysteinaemia and the severity of coronary heart disease (CHD) in Tunisian patients. Methods The study population included 100 patients with CHD and 120 healthy controls. The severity of CHD was expressed as the number of affected vessels. Superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity and total antioxidant status (TAS) concentrations were measured using commercially available methods. Plasma total homocysteine (tHcy) concentration was determined by direct chemiluminescence assay. Serum zinc (Zn) was measured by a colorimetric method. Results Compared with healthy control subjects, patients with CHD had significantly lower activities of SOD (P < 0.01), GPx (P < 0.001), and serum Zn concentrations (P < 0.001) and significantly higher tHcy concentration (P < 0.001). However TAS concentrations were not significantly different between the groups. SOD and GPx activities were negatively correlated with tHcy concentration (P < 0.05, P < 0.001, respectively). Patients with hyperhomocysteinaemia showed a lower GPx and SOD activities than patients with normohomocysteinaemia. Antioxidant enzyme activities tended to be decreased in CHD patients presenting with 0- to 3-vessel stenosis. Conclusions This study indicates that low activity of GPx, SOD and Zn concentration are associated with CHD patients. We hypothesize that hyperhomocysteinaemia and low antioxidant enzyme activities may increase the extent of CHD.

Preliminary Study of the Influence of Cubic C60 on Cultured Human Monocytes: Lack of Interleukin-1 β Secretion

Abstract Cultured human monocytes have been incubated with powdered cubic [60] fullerene. No modification has been observed with [60] fullerene when compared with negative control (monocytes alone).

In vivo reaction between [60] fullerene and vitamin A in mouse liver

An invivo biotransformation of [60] fullerene is observed that does not follow a usual redox metabolic pathway. Following the administration of a single dose of micronized C60 to Swiss mice, C60-retinol and retinyl palmitate adducts were identified in liver by UV/VIS spectroscopy and mass spectrometry after high performance liquid chromatography. NMR investigations of the main biotransformed compound, after invitro synthesis, show cycloaddition of retinol to C60. The observed biotransformation, which proves that C60 does not remain unchanged in the liver as believed previously, also shows that Diels–Alder-like reactions can occur invivo.