Laurence Nicod

Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells

The skin is the first physiological barrier, with a complex constitution, that provides defensive functions against multiple physical and chemical aggressions. Glyphosate is an extensively used herbicide that has been shown to increase the risk of cancer. Moreover there is increasing evidence suggesting that the mechanical phenotype plays an important role in malignant transformation. Atomic force microscopy (AFM) has emerged within the last decade as a powerful tool for providing a nanometer-scale resolution imaging of biological samples. Peak Force Tapping (PFT) is a newly released AFM-based investigation technique allowing extraction of chemical and mechanical properties from a wide range of samples at a relatively high speed and a high resolution. The present work uses the PFT technology to investigate HaCaT keratinocytes, a human epidermal cell line, and offers an original approach to study chemically-induced changes in the cellular mechanical properties under near-physiological conditions. These experiments indicate glyphosate induces cell membrane stiffening, and the appearance of cytoskeleton structures at a subcellular level, for low cytotoxic concentrations whereas cells exposed to IC50 (inhibitory concentration 50%) treatment exhibit control-like mechanical behavior despite obvious membrane damages. Quercetin, a well-known antioxidant, reverses the glyphosate-induced mechanical phenotype.

Glyphosate-induced antioxidant imbalance in HaCaT: The protective effect of Vitamins C and E

Roundup 3 plus(®), a glyphosate-based herbicide, is widely used in the ground, but its extensive use has posed a health risk in man. The aim of this study was firstly to investigate how glyphosate alone or included in Roundup 3 plus(®) affected the antioxidant defense system and lipid peroxidation of human cutaneous cells, and secondly, to evaluate the ameliorating effects of antioxidants, as Vitamin C (VitC) and Vitamin E (VitE), against Roundup 3 plus(®)-induced epidermal antioxidant impairment. Our results showed that glyphosate alone or included in Roundup 3 plus(®), induced significant changes in cellular antioxidant status as a glutathione depletion, enzymatic (catalase, glutathione-peroxidase and superoxide dismutase) disorders, and increased lipid peroxidation. VitC or VitE supplementation increased superoxide dismutase, glutathione-reductase and -peroxidase activities and reduced lipid peroxidation in Roundup 3 plus(®)-treated keratinocytes. These in vitro data indicated that VitC and VitE might have preventive effects against deleterious cutaneous cell damage caused by Roundup 3 plus(®).

Dietary vitamin C supplementation decreases blood pressure in DOCA-salt hypertensive male Sprague Dawley rats and this is associated with increased liver oxidative stress

The effects of a vitamin C supplemented diet on blood pressure, body and liver weights, liver antioxidant status, iron and copper levels were investigated in DOCA-salt treated and untreated Sprague-Dawley (SD) male rats after 8 weeks of treatment. Vitamin C supplementation had no effect on blood pressure in SD rats but induced a significant decrease in blood pressure in DOCA-salt treated rats, the decrease being more efficient at 50 mg/kg of vitamin C than at 500 mg/kg. Hepatic lipid peroxidation and iron levels were significantly increased in DOCA-salt hypertensive rats whereas total hepatic antioxidant capacity (HAC), glutathione peroxidase (GSH-Px) and catalase (CAT) activities were decreased. Vitamin C supplementation did not affect the overall antioxidant defences of control SD rat livers. In contrast, vitamin C supplementation accentuated the DOCA-salt induced accumulation of liver iron and lipid peroxidation. This occurred without any notable aggravation in the antioxidant deficiency of vitamin C supplemented DOCA-salt treated rat livers. Our data suggest that DOCA-salt treatment induces an accumulation of iron in rat livers which is responsible for the prooxidant effect of vitamin C. The normalization of blood pressure in DOCA-salt treated rats by vitamin C supplementation appears thus independent from liver antioxidant status.

Chemical Composition and in vitro Cytotoxic Activity of Essential Oils from Two Tropical Lamiaceae: Aeollanthus pubescens Benth. and Ocimum gratissimum L

Abstract Essential oils of Aeollanthus pubescens Benth. and Ocimum gratissimum L. (Lamiaceae) from Togo were investigated for their percentage composition and in vitro cytotoxicity. The GC and GC-MS analyses indicated that the major constituents of both essential oils were thymol, p-cymene and γ-terpinene. Testing of these volatile oils and their major constituents from commercial origin in vitro for possible cytotoxicity on the human epidermic cell line HaCat showed that the toxicity of the essential oil of A. pubescens (IC50: 1800µg.ml−1) was higher than that of the essential oil of O. gratissimum (IC50: 2400 µg.ml−1). Pure commercial thymol standard showed a cytotoxicity (IC50: 1800 µg.ml−1) identical to that of the A. pubescens essential oil. Conversely, p-cymene and γ-terpinene standards were found almost non-toxic (IC50 >3000 µg.ml−1). These findings support the assumption that the cytotoxic activities of the tested essential oils were basically due to their high level content in thymol.

Role of the magnesium cation on antihypertensive molecule-human serum albumin binding: affinity chromatography approach.

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.

Chiral discrimination of dansyl-amino-acid enantiomers on teicoplanin phase: sucrose-perchlorate anion dependence

Abstract The chiral recognition mechanism for a series of d , l -dansyl-amino-acids (test solutes) on a teicoplanin stationary phase was investigated in reversed phase liquid chromatography (RPLC). The effect of both a surface tension modifier (sucrose) and a chaotropic agent (perchlorate anion) on the enantiomeric separation was studied by varying their concentration, c , in the mobile phase. The thermodynamic data supported the fact that the sucrose molecule acted only on the hydrophobic part of the interaction teicoplanin/dansyl-amino-acid and not on the specific chiral part. It was demonstrated that the enhancement of the separation factor observed as the perchlorate salt concentration increased in the mobile phase was enthalpically controlled owing to stereoselective bonding interactions. Such behavior was used to optimize the chromatographic conditions for separation of dansyl-amino-acids on teicoplanin.

Imidazo[2,1-b]thiazole derivatives. XI: Modulation of the CD2-receptor of human T trypsinized lymphocytes by several imidazo[2,1-b]thiazoles

Abstract About 40 substituted imidazo[2,1-b]thiazoles were obtained in order to study their in vitro immunological effect on the modulation of the expression of human T trypsinized lymphocytes by the CD2 receptor. A synthetic program was developed to introduce either an oxygenated function, such as ester (11, 14), acid (12) and arylketonic groups (9, 13, 15), or two groups, such as an aryl and an ester (1, 6, 8), an acid (3, 7) or a hydrazide (2). These compounds were examined by an E-rosette-forming-cell test, and display a positive drug efficacity index, suggesting a regeneration effect on the expression of CD2 receptors. The following structural parameters are favourable: an aryl moiety on the C-6 with a methoxy or nitro group; and an ethyl ester on the C-3, a double bond to the 2,3-position (the 5,6-position is ineffective). Acid and hydrazide functions or the loss of phenyl group on the C-6 decrease this activity. If the aryl group is on the C-3 or C-2 side chain, the activity is weaker and more so for the latter. However, the most interesting derivatives are less immunostimulating than levamisole hydrochloride.

Chemical Composition and In Vitro Cytotoxic Activity of Xylopia aethiopica (Dun) A. Rich. (Annonaceae) Fruit Essential Oil from Togo

Abstract Essential oil extracted (4.4% in yield) from air-dried fruits of Xylopia aethiopica harvested in Togo was investigated for percentage composition and in vitro cytotoxicity. The chemical composition of the essential oil was examined by GC and GC/MS. Thirty-five compounds were identified representing 89.9% of total oil. The major constituents were β-pinene (23.6%), α-pinene (11%), sabinene (9.8%), germacrene D (8.3%) and 1,8 cineole (8.2%). The cytotoxicity of the volatile oil was evaluated in vitro on the human epidermal cell line HaCaT. The tested sample did not show any cytotoxicity (IC50 >3000 μg.ml-1) effect at concentrations around 3000 μg.ml-1. Further testing in bioassay would probably help in validating some of medicinal uses of X. aethiopica in topical drugs and/or in cosmetics as natural products.

Supercoiled circular DNA and protein retention in non-equilibrium chromatography: Temperature and velocity dependence: testimony of a transition

Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C1 column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 degrees C) the plasmid retention increased over the entire flow-rate range (0.02-1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.

From surface to intracellular non-invasive nanoscale study of living cells impairments

Among the enduring challenges in nanoscience, subsurface characterization of living cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale. However, measurements in liquid environments remain complex, in particular in the subsurface domain. Here we introduce liquid-mode synthesizing atomic force microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach, l-MSAFM, for nanoscale imaging of living cell in their physiological environment or in presence of a chemical stress agent could resolve the loss of inner structures induced by glyphosate, the main component of a well-known pesticide (RoundUp™). This firsthand ability to monitor the cells inner response to external stimuli non-destructively and in liquid, has the potential to unveil critical nanoscale mechanisms of life science.