Morphological damages of a glyphosate-treated human keratinocyte cell line revealed by a micro- to nanoscale microscopic investigation.

Among the molecules to which the human skin is exposed, glyphosate is used as an herbicide. Glyphosate has been shown to induce in vitro cutaneous cytotoxic effects, concomitant with oxidative disorders. In this following study, we focused on dynamic events of the loss of HaCaT cell integrity appearing after a glyphosate treatment. In these conditions, we showed that glyphosate is able to disrupt HaCaT cells and to induce intracellular oxidative cascade. In this aim, we optimized the conditions of cell treatment playing on exposure time (from 24 h to 30 min), which directly modify the cell viability profile (glyphosate 50% inhibition concentration from 28 to 53 mM) and allow to track cells along the treatment as an "induction and visualization" process. The combination of atomic force and fluorescence microscopic approaches offered opportunities to lead in parallel an investigation of the membrane surface and of the intracellular disorders, through cytoskeleton, nuclear, and oxidative stress marker targeting. The originality of our approach relies on monitoring all events derived from oxidative stress in process and performed by simultaneous cytotoxic induction and nanoscale cell visualization. We revealed a transition from spread and globular to elongated cell morphology, with a drastic cell size reduction, after a dose- and time-dependent glyphosate treatment; a redistribution of cell surface protrusions was also pointed out. All these membrane damages, added to observations of disorganized cytoskeleton, condensed chromatin, and overproduction of oxidative reactive species, lead us to conclude that glyphosate acts in induction of apoptotic process.

Synthesis and antioxidant activity evaluation of new hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones obtained by Biginelli reaction in two steps.

New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-carboxylates, previously prepared by Biginelli reaction using appropriate aldehyde, urea derivatives and ethyl acetoacetate. Their antioxidant properties were evaluated by two methods: scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and scavenging effect on hydroxyl radicals. The results show that the compounds containing thiourea moiety have better activity.

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(®).

Association mechanism between a series of rodenticide and humic acid: a frontal analysis to support the biological data.

The binding constants (K) of a series of anticoagulant rodenticides with the main soil organic component, humic acid (HA), were determined using frontal analysis approach. The order of the binding constants was identical as the one obtained in a previous paper [J. Chromatogr. B 813 (2004) 295], i.e. bromadiolone>brodifacoum>difenacoum>chlorophacinone>diphacinone, confirming the power of this frontal analysis approach for the determination of binding constants. Moreover, and for the first time, the concentration of unbound rodenticide to HAs could be determined. Thanks this approach, we could clearly demonstrate that HA acid protected the human hepatoma cell line HepG2 against the cytotoxicity of all the rodenticides tested and that the toxicity of rodenticides was directly linked to the free rodenticide fraction in the medium (i.e. unbound rodenticide to HA).

Vitamins C and E reverse effect of herbicide-induced toxicity on human epidermal cells HaCaT: a biochemometric approach.

The purpose of this study was to investigate and compare the cytotoxicity of glyphosate alone or included in Roundup 3 plus modulated by the cytoprotective effects of additional antioxidants such as Vitamin C and Vitamin E on the human keratinocytes cell line HaCaT. An experimental design which allows to minimize the number of experiments was carried out to determine the optimal conditions for cytoprotection against herbicide-induced toxicity. It was shown that HaCaT cell line provides a useful model to study components with toxicity or antioxidant activity. Our results indicated that (i) glyphosate-based formulations can be responsible for oxidative damage to human epidermal cells, (ii) antioxidant compounds should be associated to herbicide formulations to decrease their deleterious effects on human skin. The use of an experimental design connected with the simplex method can be consider to be a fast technique to classify, with a limited number of experiments, the respective role of five parameters in the in vitro cytoprotection by antioxidants of herbicide-induced toxicity.

Evolution of liver antioxidant status and iron implication during the development of deoxycorticosterone-saline hypertension in rats.

Hypertension is known to be associated with an oxidative stress resulting from an imbalance of antioxidant defense mechanisms in various tissues. The purpose of this study was to investigate the relationship between the increase of arterial blood pressure, measured during the gradual development of experimental hypertension in deoxycorticosterone (DOCA)-salt-treated rats, and an early imbalance of liver antioxidant status. The levels of liver oxidant/antioxidant markers and iron were studied during the induction of hypertension in 3-, 6-, and 8-wk DOCA-salt-treated Sprague-Dawley rats. Hepatic antioxidant defenses were decreased as early as 3 wk of hypertensive treatment: the decrease of peroxidase-reductase-transferase and catalase activities was associated with a significant increase of thiobarbituric acid reactive substances (TBARS) levels. Liver oxidative stress increased until 6 wk and remained stable at 8 wk of DOCA-salt treatment. Concurrently, liver iron levels were increased at 6 wk and returned to normal values after 8 wk of hypertensive treatment. Iron seems to be an inductor of liver oxidative stress and responsible for the persistent oxidative stress, most likely through secondary free-radical release. Thus, our data (1) confirm that hypertension in DOCA-salt-treated rats might be a free-radical-dependent disease where hepatic oxidant/antioxidant imbalance is obviously involved from the beginning of blood pressure elevation and (2) suggest that the use of suitable iron chelators might reverse liver oxidative stress associated with the increase of blood pressure.

Rodenticide-humic acid adsorption mechanisms and role of humic acid on their toxicity on human keratinocytes: chromatographic approach to support the biological data.

Humic substances are the most important soil components affecting the behaviour and performances of herbicides in the soil-water-organism system. In this paper, a chromatographic approach was used for analysis of anticoagulant rodenticide-humic acid adsorption mechanisms. Using an equilibrium perturbation method, it was clearly shown that: (i) humic acid can be adsorbed on the C18 stationary phase, and (ii) all the rodenticides can be adsorbed on the humic acid adsorbed on the C18 stationary phase. This approach allowed the determination of the adsorption constant values between the anticoagulant rodenticides and humic acid as well as the corresponding thermodynamic data of this adsorption mechanism. The role of humic acid on the toxicity of these rodenticides on human keratinocytes was also clearly described in relation to these physico-chemical data.

Synthesis of 4-hydroxycoumarin and 2,4-quinolinediol derivatives and evaluation of their effects on the viability of HepG2 cells and human hepatocytes culture.

We report here the synthesis of aromatic coumarins and aromatic alpha-quinolones which were evaluated in vitro for their protective potentialities against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage on human liver cell death, i.e., human hepatoma HepG2 cell line and human hepatocytes in primary culture. We found that the presence of a benzylidene at the 3-position or a heterocycle with N and S heteroatoms on the benzopyrone or quinolone system was essential for the protective effect of these compounds against t-BHP-induced decrease in viability of cells. We found also that a methoxy group on the aromatic ring systems decreased this potential. t-BHP-induced cytotoxicity in primary cultures of human hepatocytes could be therefore prevented by these compounds suggesting that they could display hepatoprotective effects in humans.

A biochromatographic framework to evaluate the calcium effect on the antihypertensive molecule-human serum albumin binding.

The Ca(2+) cation effect on the antihypertensive molecule binding on human serum albumin (HSA) was studied by biochromatography. The thermodynamic parameters corresponding to this binding were determined for a wide range of Ca(2+) concentration (x). For the two antihypertensive molecules under study, their binding to HSA can be divided into two Ca(2+) cation concentration regions due to a HSA phase transition. This result was confirmed by an enthalpy-entropy investigation. For a low x value (below x(c)=1.6 mmol l(-1)), the HSA cavity was in an ordered solid-like state leading to an increase in the interactions between the antihypertensive drugs and the HSA cavity and consequently, a solute-HSA affinity increase. For x above x(c), the HSA cavity was in a disordered solid-like state, implying a decrease in the antihypertensive drug-HSA binding.

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.