Pierre Stocker

Rigid Orthogonal bis-TEMPO Biradicals with Improved Solubility for Dynamic Nuclear Polarization

The synthesis and characterization of oxidized bis-thioketal-trispiro dinitroxide biradicals that orient the nitroxides in a rigid, approximately orthogonal geometry are reported. The biradicals show better performance as polarizing agents in dynamic nuclear polarization (DNP) NMR experiments as compared to biradicals lacking the constrained geometry. In addition, the biradicals display improved solubility in aqueous media due to the presence of polar sulfoxides. The results suggest that the orientation of the radicals is not dramatically affected by the oxidation state of the sulfur atoms in the biradical, and we conclude that a biradical polarizing agent containing a mixture of oxidation states can be used for improved solubility without a loss in performance.

Isolation and characterization of a new steroid derivative as a powerful antioxidant from Cleome arabica in screening the in vitro antioxidant capacity of 18 Algerian medicinal plants.

Hydromethanolic extracts from 18 Algerian medicinal plants were screened for their phenolic contents and radical scavenging activities. The phenolic extract of Cleome arabica (Capparaceae) was found to be the most active one. Purification of this extract by semi-preparative high performance liquid chromatography led to the isolation and identification of new steroid derivative. The structure of the active principle is proposed as (17-(4-hydroxy-1,5-dimethylhexyl)-2,3,7-(acetyloxy) gona-1,3,5(10)-trien-15-ol). Compared to six other standard antioxidants which were ascorbic acid, α-tocopherol, Trolox, (+) catechin, p-coumaric acid and gallic acid, the isolated compound was found to be significantly more active in the radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Similar results were obtained in the hemolysis assay. The antioxidant capacities of the methanolic extract of C. arabica and its principle compound indicate that this plant may be an important source of chemopreventive and chemotherapeutic natural products activity.

Effect of reactive oxygen and carbonyl species on crucial cellular antioxidant enzymes

Numerous reactive oxygen species (ROS) and reactive carbonyl species (RCS) issuing from lipid and sugar oxidation are known to damage a large number of proteins leading to enzyme inhibition and alteration of cellular functions. Whereas studies in literature only focus on the reactivity of one or two of these compounds, we aimed at comparing in the same conditions of incubations (4 and 24h at 37°C) the effects of both various RCS (4-hydroxynonenal, 4-hydroxyhexenal, acrolein, methylglyoxal, glyoxal, malondialdehyde) and ROS (H₂O₂, AAPH) on the activity of key enzymes involved in cellular oxidative stress: superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH). This was realized both in vitro on purified proteins and MIAPaCa-2 cells. Incubation of these enzymes with RCS resulted in a significant time- and concentration-dependent inhibition for both pure enzymes and in cell lysates. Among all RCS and ROS, hydroxynonenal (HNE) was observed as the most toxic for all studied enzymes except for SOD and is followed by hydrogen peroxide. At 100μM, HNE resulted in a 50% reduction of GPx, 56% of GST, 65% of G6PDH, and only 10% of Cu,Zn-SOD. Meanwhile it seems that concentrations used in our study are closer to biological conditions for ROS than for RCS. H₂O₂ and AAPH-induced peroxyl radicals may be probably more toxic towards the studied enzymes in vivo.

DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity

Abstract The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 × 10−5–6 × 10−3 g/l corresponding to a concentration range of 0.22–22 µM) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.

Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.

The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury.

Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase

Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.

Fluorimetric screening assay for protein carbonyl evaluation in biological samples

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.

High throughput assay for evaluation of reactive carbonyl scavenging capacity.

Many carbonyl species from either lipid peroxidation or glycoxidation are extremely reactive and can disrupt the function of proteins and enzymes. 4-hydroxynonenal and methylglyoxal are the most abundant and toxic lipid-derived reactive carbonyl species. The presence of these toxics leads to carbonyl stress and cause a significant amount of macromolecular damages in several diseases. Much evidence indicates trapping of reactive carbonyl intermediates may be a useful strategy for inhibiting or decreasing carbonyl stress-associated pathologies. There is no rapid and convenient analytical method available for the assessment of direct carbonyl scavenging capacity, and a very limited number of carbonyl scavengers have been identified to date, their therapeutic potential being highlighted only recently. In this context, we have developed a new and rapid sensitive fluorimetric method for the assessment of reactive carbonyl scavengers without involvement glycoxidation systems. Efficacy of various thiol- and non-thiol-carbonyl scavenger pharmacophores was tested both using this screening assay adapted to 96-well microplates and in cultured cells. The scavenging effects on the formation of Advanced Glycation End-product of Bovine Serum Albumin formed with methylglyoxal, 4-hydroxynonenal and glucose-glycated as molecular models were also examined. Low molecular mass thiols with an α-amino-β-mercaptoethane structure showed the highest degree of inhibitory activity toward both α,β-unsaturated aldehydes and dicarbonyls. Cysteine and cysteamine have the best scavenging ability toward methylglyoxal. WR-1065 which is currently approved for clinical use as a protective agent against radiation and renal toxicity was identified as the best inhibitor of 4-hydroxynonenal.

A nonradioisotope chemiluminescent assay for evaluation of 2-deoxyglucose uptake in 3T3-L1 adipocytes. Effect of various carbonyls species on insulin action.

We have developed a rapid nonradioisotope chemiluminescent assay adapted to high-throughput screening experiments, to evaluate glucose uptake activity in cultured cells. For chemiluminescence quantification of 2-deoxyglucose, we used a luminol oxidation reaction after an enzymatic dephosphorylation of 2-deoxyglucose-6-phosphate. All reactions were performed at 37 °C by consecutive addition of reagents, and the assay is able to quantify 2DG in picomole per well. To confirm the reliability of this method, we have evaluated the dose-effect of insulin, GLUT4 inhibitors and insulin-sensitizing agent on 2DG uptake into 3T3-L1 cells. The results obtained with the assay for 2DG uptake in vitro in the absence or presence of insulin stimulation, were similar to those obtained by the previous radioisotopic and enzymatic methods. We have also used this assay to evaluate the effect of various reactive carbonyl and oxygen species on insulin-stimulated 2DG-uptake into adipocytes. All reactive carbonyl species tested decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner without affecting basal glucose uptake in 3T3-L1 cells. 4-hydroxynonenal was found to be the most potent in the impairment of glucose uptake. This new enzymatic chemiluminescent assay is rapid and useful for measurement of 2DG uptake in insulin-responsive in cultured cells.

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin-mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure-function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.