Carolina Soledad Martinez

Hepatocellular alterations and dysregulation of oncogenic pathways in the liver of transgenic mice overexpressing growth hormone.

Growth hormone (GH) overexpression throughout life in transgenic mice is associated with the development of liver tumors at old ages. The preneoplastic pathology observed in the liver of young adult GH-overexpressing mice is similar to that present in humans at high risk of hepatic cancer. To elucidate the molecular pathogenesis underlying the pro-oncogenic liver pathology induced by prolonged exposure to elevated GH levels, the activation and expression of several components of signal transduction pathways that have been implicated in hepatocellular carcinogenesis were evaluated in the liver of young adult GH-transgenic mice. In addition, males and females were analyzed in parallel in order to evaluate sexual dimorphism. Transgenic mice from both sexes exhibited hepatocyte hypertrophy with enlarged nuclear size and exacerbated hepatocellular proliferation, which were higher in males. Dysregulation of several oncogenic pathways was observed in the liver of GH-overexpressing transgenic mice. Many signaling mediators and effectors were upregulated in transgenic mice compared with normal controls, including Akt2, NFκB, GSK3β, β-catenin, cyclin D1, cyclin E, c-myc, c-jun and c-fos. The molecular alterations described did not exhibit sexual dimorphism in transgenic mice except for higher gene expression and nuclear localization of cyclin D1 in males. We conclude that prolonged exposure to GH induces in the liver alterations in signaling pathways involved in cell growth, proliferation and survival that resemble those found in many human tumors.

Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver.

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling. EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.

Relation between biophysical properties of nanostructures and their toxicity on zebrafish

In recent years, the use of commercial nanoparticles in different industry and health fields has increased exponentially. However, the uncontrolled application of nanoparticles might present a potential risk to the environment and health. Toxicity of these nanoparticles is usually evaluated by a fast screening assay in zebrafish (Danio rerio). The use of this vertebrate animal model has grown due to its small size, great adaptability, high fertilization rate and fast external development of transparent embryos. In this review, we describe the toxicity of different micro- and nanoparticles (carbon nanotubes, dendrimers, emulsions, liposomes, metal nanoparticles, and solid lipid nanoparticles) associated to their biophysical properties using this model. The main biophysical properties studied are size, charge and surface potential due to their impact on the environment and health effects. The review also discusses the correlation of the effects of the different nanoparticles on zebrafish. Special focus is made on morphological abnormalities, altered development and abnormal behavior. The last part of the review debates changes that should be made in future directions in order to improve the use of the zebrafish model to assess nanotoxicity.

In vivo study of teratogenic and anticonvulsant effects of antiepileptics drugs in zebrafish embryo and larvae

Epilepsy is a neurological disorder treated with antiepileptic drugs (AEDs). Since AEDs are administered in women in childbearing age, it is critical to study if drugs are capable of inducing developmental toxicity. Along the bibliography available, there is no research comparing teratogenicity and anticonvulsant effect within the same study. In the present study, we evaluated the teratogenic and anticonvulsant effects of six different AEDs: carbamazepine, levetiracetam, lamotrigine, phenobarbital, phenytoin and valproic acid. Zebrafish was the selected animal model because of its small size, rapid external development and similar neurophysiology to mammals. Zebrafish embryo and larvae were exposed to AEDs. Embryo development was monitored by their hatching and morphology. In larvae, locomotor activity was measured as a parameter of neurotoxicity. Finally, anticonvulsant effect was determined after exposure to AEDs in zebrafish larvae treated with the proconvulsant drug pentylenetetrazole. Our results suggest that lamotrigine and phenytoin could be suitable non-teratogenic and efficient anticonvulsant options for epilepsy treatment.

PAMAM dendrimers as a carbamazepine delivery system for neurodegenerative diseases: A biophysical and nanotoxicological characterization

Graphical abstract Figure. No Caption available. HighlightsDG4.0 and DG4.5 encapsulated around of 20 molecules of CBZ per dendrimer.DG4.0‐CBZ and DG4.5‐CBZ were stable for 90 days and resistant to lyophilization.Both DG4.0‐CBZ and DG4.5‐CBZ complexes showed a controlled release of the CBZ.DG4.5‐CBZ were biocompatible according to the studies on zebrafish and cell culture.A stable and biocompatible drug delivery system (DG4.5‐CBZ) has been developed. Abstract Carbamazepine (CBZ) is an antiepileptic drug, which also could be used in the treatment of neurodegenerative diseases, such as the Alzheimers disease. However, its use has been limited due to its low solubility, inefficient pharmacokinetic profiles, and multiple side effects. PAMAM dendrimers, ethylenediamine core, generation 4.0 (amine terminal groups) and 4.5 (carboxylate terminal groups) (DG4.0 and DG4.5 respectively) are polymers that can increase drug solubility through complexation. Thus, the aim of this work was to obtain and characterize complexes between CBZ and dendrimers. Both DG4.0 and DG4.5 allowed the incorporation of ˜20 molecules of CBZ per dendrimer, into their hydrophobic pockets. DG4.0‐CBZ and DG4.5‐CBZ complexes were found to be stable for 90 days at 37 °C and resistant to a lyophilization process, presenting controlled drug release. Also, the complexes nanotoxicity was tested ex vivo (human red blood cells), in vitro (N2a cell line), and in vivo (zebrafish). No hemolytic effect was observed in the ex vivo model. As regards in vitro toxicity, the DG4.5‐CBZ complexes significantly reduced the toxicity caused by the free drug. Moreover, the DG4.5‐CBZ did not cause neurotoxicity or cardiotoxicity in zebrafish larvae. In conclusion, a stable and biocompatible drug delivery system based on the DG4.5 capable of complex the CBZ has been developed. This achievement highlights the advantages of using negatively charged dendrimers for nanomedicine.

Folic acid magnetic nanotheranostics for delivering doxorubicin: Toxicological and biocompatibility studies on Zebrafish embryo and larvae

ABSTRACT Doxorubicin (DOXO) is a chemotherapeutic agent widely used for the treatment of solid tumors and hematologic malignancies in both adults and children. However, DOXO causes short‐ and long‐term cardiotoxicity and others undesirable side effects, such as nephrotoxicity and neurotoxicity. Magnetic nanoparticles (MNPs) allow the delivery of drugs specifically to target place, employing an external magnet. Moreover, they may act as contrast agents in MRI providing information on the diagnostic of diverse pathologies. In this way, two functions may be combined in a unique nanosystem known as theranostic. Also, the MNPs can be modified with folic acid (MNPs@FA) to increase the uptake by cancer cells that overexpress the FA receptors. In previous works, our collaborators obtained and characterized MNPs, MNPs@FA, and MNPs@FA@DOXO. It is essential to study the biosafety of nanotheranostic, and there is no published study of Fe3O4 nanoparticles developmental toxicity. Because of that, this work aimed to study the in vivo toxicity and biocompatibility of DOXO, MNPs@FA, and MNPs@FA@DOXO using zebrafish embryo and larvae as an animal model. Viability, developmental toxicity, changes in spontaneous movement (neurotoxicity), changes in cardiac rhythm (cardiotoxicity), and efficiency of DOXO‐uptake were studied. While the 48‐h treatment with 50 &mgr;g/mL of DOXO resulted in a 30% larvae death and the development of significant morphological abnormalities, the treatment with MNPs@FA@DOXO and MNPs@FA did not reduce the viability and did not cause developmental abnormalities. Besides, the MNPs@FA@DOXO reduced the cardiotoxicity and promoted a more rapid and significant uptake of DOXO by zebrafish larvae. Graphical Abstract Figure. No Caption available. HighlightsThis work is the first studying the toxicity of Fe3O4 nanoparticles in zebrafish.Doxorubicin showed to be toxic in both zebrafish embryo and larvae.Folic acid magnetic nanotheranostic reduced the toxicity caused by Doxorubicin.Folic acid magnetic nanotheranostic proved to be a biocompatible delivery system.The developed nanotheranostic improved the Doxorubicin‐uptake at shorter times.