Minerva Martínez-Alfaro

Effect of melatonin administration on DNA damage and repair responses in lymphocytes of rats subchronically exposed to lead.

Lead exposure induces DNA damage, oxidative stress, and apoptosis, and alters DNA repair. We investigated the effects of melatonin co-administered to rats during exposure to lead. Three doses of lead acetate (10, 50 and 100mg/kg/day) were administered to rats during a 6-week period. Lymphocytes were analyzed. Lead exposure decreased glutathione (GSH) levels in blood, and at doses of 100mg/kg/day and 50mg/kg/day without melatonin, caused high levels of DNA damage, induced apoptosis, and altered DNA repair. Melatonin co-treatment did not attenuate the effects of lead at 100mg/kg/day, indicating that the effect of melatonin on GSH reduction is not sufficient to reduce the genotoxic effects of lead at this high dose. After 6 weeks of treatment, decreased weight gain was observed in high lead-dose groups (100mg/kg/day), with or without melatonin, and in medium-dose groups (50mg/kg/day) with melatonin, compared with the control group. The protective action of melatonin against lead toxicity is dependent on the dose of lead. Further pharmacological studies are needed to determine whether melatonin acts via melatonin membrane receptors on lymphocytes.

Melatonin attenuates the effects of sub-acute administration of lead on kidneys in rats without altering the lead-induced reduction in nitric oxide

Exposure to lead induces oxidative stress and renal damage. Although most forms of oxidative stress are characterized by simultaneous elevation of nitrogen and oxidative species, lead-induced oxidative stress is unusual in that it is associated with a reduction in nitric oxide (NO) levels in the kidney. The role of NO in kidney injury is controversial; some studies suggest that it is associated with renal injury, whereas others show that it exerts protective effects. Concentration-dependent effects have also been proposed, linking low levels with vasodilatation and high levels with toxicity. The aim of this study was to evaluate the effects of melatonin co-exposure on the lead-induced reduction in renal NO levels. We found that sub-acute intraperitoneal administration of 10 mg/kg/day of lead for 15 days induced toxic levels of lead in the blood and caused renal toxicity (pathological and functional). Under our experimental conditions, lead induced an increase in lipid peroxidation and a decrease in NO. Melatonin co-treatment decreased lead-induced oxidative stress (peroxidation level) and toxic effects on kidneys without altering the lead-induced reduction in renal NO. These results suggest that, in our experimental model, the reduction in renal NO levels by lead exposure is not the only responsible factor for lead-induced kidney damage.

Oxidative stress effects of thinner inhalation

Thinners are chemical mixtures used as industrial solvents. Humans can come into contact with thinner by occupational exposure or by intentional inhalation abuse. Thinner sniffing causes damage to the brain, kidney, liver, lung, and reproductive system. We discuss some proposed mechanism by which thinner induces damage. Recently, the induction of oxidative stress has been suggested as a possible mechanism of damage. This paper reviews the current evidence for oxidative stress effects induced by thinner inhalation. Early ideas about the effects of thinner on lipids are discussed in one section. We discuss several studies that have shown the oxidative effects of thinner inhalation on: lipid peroxidation, levels of antioxidant enzymes, glutathione depletion, and oxidation of proteins and DNA. We have also included studies about oxidative stress effects induced by toluene, the principal component (60-70%) of thinner. Finally, work describing the effects of oxidative stress induced by thinner inhalation on different organs is discussed.

Functionalization and characterization of persistent luminescence nanoparticles by dynamic light scattering, laser Doppler and capillary electrophoresis.

Zinc gallate nanoparticles doped with chromium (III) (ZnGa1.995O4:Cr0.005) are innovative persistent luminescence materials with particular optical properties allowing their use for in vivo imaging. They can be excited in the tissue transparency window by visible photons and emit light for hours after the end of the excitation. This allows to observe the probe without any time constraints and without autofluorescence signals produced by biological tissues. Modification of the surface of these nanoparticles is essential to be colloidally stable not only for cell targeting applications but also for proper distribution in living organisms. The use of different methods for controlling and characterizing the functionalization process is imperative to better understand the subsequent interactions with biological elements. This work explores for the first time the characterization and optimization of a classic functionalization sequence, starting with hydroxyl groups (ZGO-OH) at the nanoparticle surface, followed by an aminosilane-functionalization intermediate stage (ZGO-NH2) before PEGylation (ZGO-PEG). Dynamic light scattering and laser doppler electrophoresis were used in combination with capillary electrophoresis to characterize the nanoparticle functionalization processes and control their colloidal and chemical stability. The hydrodynamic diameter, zeta potential, electrophoretic mobility, stability over time and aggregation state of persistent luminescence nanoparticles under physiological-based solution conditions have been studied for each functional state. Additionally, a new protocol to improve ZGO-NH2 stability based on a thermal treatment to complete covalent binding of (3-aminopropyl) triethoxysilane onto the particle surface has been optimized. This thorough control increases our knowledge on these nanoparticles for subsequent toxicological studies and ultimately medical application.

Melatonin reduces lead levels in blood, brain and bone and increases lead excretion in rats subjected to subacute lead treatment.

Melatonin, a hormone known for its effects on free radical scavenging and antioxidant activity, can reduce lead toxicity in vivo and in vitro.We examined the effects of melatonin on lead bio-distribution. Rats were intraperitoneally injected with lead acetate (10, 15 or 20mg/kg/day) with or without melatonin (10mg/kg/day) daily for 10 days. In rats intoxicated with the highest lead doses, those treated with melatonin had lower lead levels in blood and higher levels in urine and feces than those treated with lead alone, suggesting that melatonin increases lead excretion. To explore the mechanism underlying this effect, we first assessed whether lead/melatonin complexes were formed directly. Electronic density functional (DFT) calculations showed that a lead/melatonin complex is energetically feasible; however, UV spectroscopy and NMR analysis showed no evidence of such complexes. Next, we examined the liver mRNA levels of metallothioneins (MT) 1 and 2. Melatonin cotreatment increased the MT2 mRNA expression in the liver of rats that received the highest doses of lead. The potential effects of MTs on the tissue distribution and excretion of lead are not well understood. This is the first report to suggest that melatonin directly affects lead levels in organisms exposed to subacute lead intoxication.

Photo-stimulation of persistent luminescence nanoparticles enhances cancer cells death

Persistent luminescence nanoparticles made of ZnGa1.995Cr0.005O4 (ZGO-NPs) are innovative nanomaterials that emit photons during long periods of time after the end of the excitation, allowing their use as diagnosis probes for in vivo optical imaging. During the excitation process, a part of the energy is stored in traps to further emit photons over long time. However, we observed in this study that some of the energy reduces molecular oxygen to produce reactive oxygen species (ROS). Following this observation, theoxidative stress induction and cytotoxic effects of these NPs were investigated on human breast cancer cells. The results indicate that ROS production was stimulated by exposition of the hydroxylated ZGO-NPs to UV or visible light, and the oxidative stress induced in cells after internalization can be directly correlated to their dose-dependent inhibition of cell viability. On the contrary, PEGylated ZGONPs were not uptaken by cells and have no effect on the production of ROS. Thus, the cell viability was not altered by these nanoparticles. This study reveals the importance of considering light irradiation and surface coating of luminescent nanoparticles toxicity which open new perspectives for their use in photodynamic therapy.

Electrokinetic Hummel-Dreyer characterization of nanoparticle-plasma protein corona: The non-specific interactions between PEG-modified persistent luminescence nanoparticles and albumin

Nanoparticles (NPs) play an increasingly important role in the development of new biosensors, contrast agents for biomedical imaging and targeted therapy vectors thanks to their unique properties as well as their good detection sensitivity. However, a current challenge in developing such NPs is to ensure their biocompatibility, biodistribution, bioreactivity and in vivo stability. In the biomedical field, the adsorption of plasmatic proteins on the surface of NPs impacts on their circulation time in blood, degradation, biodistribution, accessibility, the efficiency of possible targeting agents on their surface, and their cellular uptake. NP surface passivation is therefore a very crucial challenge in biomedicine. We developed herein for the first time an electrokinetic Hummel-Dreyer method to quantitatively characterize the formation of protein corona on the surface of NPs. This strategy was designed and optimized to evaluate the non specific binding of bovine serum albumin with the recently discovered PEG-functionalized ZnGa1.995Cr0.005O4 persistent luminescence NPs developed for in vivo biological imaging. The binding strength and the number of binding sites were determined at different ionic strengths. This methodology opens the way to an easy, low sample- and low time-consuming evaluation of the impact of NP surface modification on protein-corona formation and therefore on their potential for various bio-medical applications.

Simultaneous Electrochemical Speciation of Oxidized and Reduced Glutathione. Redox Profiling of Oxidative Stress in Biological Fluids with a Modified Carbon Electrode

The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNTf) was developed to modify glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode toward reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNTf results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry reveals detection limits of 100 μM for GSH and 8.3 μM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrixes without any pretreatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication.

Subsurface cistern (SSC) proliferation in Purkinje cells of the rat cerebellum in response to acute and chronic exposure to paint thinner: A light and electron microscopy study

Intentional inhalation and occupational exposure are two ways humans are exposed to thinner, a widely employed solvent in industry. Inhalation of thinner induces toxic effects in various organs, with the cerebellum being one of the most affected structures of the CNS. The aim of this work was to describe specific structural alterations of cerebellum Purkinje cells in rats following exposure to thinner for 16 weeks. A histological analysis of the cerebellum of solvent-exposed rats revealed swollen Purkinje cell dendrites surrounded by empty space, and electronic microscopy showed an increase in the number of subsurface cisterns (SSCs) within their dendritic processes. After a period of non-exposure, the number of SSCs decreased without reaching normal levels, suggesting a degree of plasticity. Purkinje cell SSCs, which are derived from smooth endoplasmic reticulum, contain inositol trisphosphate receptors (IP3Rs), ryanodine receptors (RR), and a recently identified characteristic cluster of large conductance calcium-activated potassium (BKCa) channels. We found that SSCs in Purkinje cell dendrites were closely associated with mitochondria, and immunofluorescence microscopy showed higher levels of RR and calbindin receptors (CB), in Purkinje cells of exposed than normal rats. These changes are probably related to behavioral manifestations of cerebellar alterations, such as imbalance and ataxia, consistent with the suggested involvement of increases in SSCs in ataxia in rats and humans. This increase in SSCs, taken together with the localization of RR, IP3R and BKCa proteins in this structure, suggests altered intracellular calcium-buffering processes in the Purkinje cells of thinner-exposed rats.

Long-term toxicological effects of persistent luminescence nanoparticles after intravenous injection in mice

The ZnGa1.995Cr0.005O4 persistent luminescence nanoparticles offer the promise of revolutionary tools for biological imaging with applications such as cell tracking or tumor detection. They can be re-excited through living tissues by visible photons, allowing observations without any time constraints and avoiding the undesirable auto-fluorescence signals observed when fluorescent probes are used. Despite all these advantages, their uses demand extensive toxicological evaluation and control. With this purpose, mice were injected with a single intravenous administration of hydroxylated or PEGylated persistent luminescence nanoparticles at different concentrations and then a set of standard tests were carried out 1day, 1 month and 6 months after the administration. High concentrations of hydroxylated nanoparticles generate structural alterations at histology level, endoplasmic reticulum damage and oxidative stress in liver, as well as rising in white blood cells counts. A mechanism involving the endoplasmic reticulum damage could be the responsible of the observed injuries in case of ZGO-OH. On the contrary, no toxicological effects related to PEGylated nanoprobes treatment were noted during our in vivo experiments, denoting the protective effect of PEG-functionalization and thereby, their potential as biocompatible in vivo diagnostic probes.